Monochrome multiplex quantitative PCR

ABSTRACT

Disclosed herein are methods and compositions for determining the copy number of a first target nucleic acid as compared to the copy number of a second target nucleic acid in a single well with a single detection label. For example, disclosed herein are methods and compositions for determining the copy number of a first target nucleic acid as compared to the copy number of a second target nucleic acid by a monochrome multiplex quantitative PCR (MMQPCR) in a single well with a single detection label.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/139,890, filed on Dec. 22, 2008, which is hereby incorporated hereinby reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

Portions of the research and inventions disclosed herein may have beenmade with U.S. Government support under the National Institutes ofHealth Grant No. 5R21AG030034. The U.S. government has certain rights inthis invention.

TECHNICAL FIELD

Disclosed are methods for determining the relative and absolute copynumber of a first target nucleic acid sequence as compared to a secondtarget nucleic acid sequence in a single reaction with a singledetection label and kits useful in practicing the methods.

BACKGROUND OF THE INVENTION

Real-time quantitative polymerase chain reaction (QPCR) determines, foreach reaction well, the C_(t), i.e. the fractional cycle number at whichthe well's rising fluorescence (proportional to product formation)crosses a set threshold that is several standard deviations above thebaseline fluorescence (Higuchi, R., Fockler, C., Dollinger, G. andWatson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNAamplification reactions. Biotechnology (N Y), 11, 1026-1030). The C_(t)versus log(amount of input target DNA) plot is linear, allowing relativequantitation of unknowns by comparison to a standard curve derived fromamplifying, in the same plate, serial dilutions of a reference DNAsample.

For many QPCR applications, the investigator wishes to normalize thesignal from a target sequence (T) to the signal from a referencesequence (R). Early studies measured T and R in separate (monoplex)reactions with a dye that fluoresces upon intercalation into anydouble-stranded DNA, e.g. ethidium bromide or SYBR Green I, and thisapproach continues. More recent studies have measured T and R in thesame reaction vessel, in a multicolor multiplex QPCR, using separatefluorescent dyes with distinct excitation/emission spectra for each ofthe DNA sequences being quantified (Wittwer, C. T., Herrmann, M. G.,Gundry, C. N. and Elenitoba-Johnson, K. S. (2001) Real-time multiplexPCR assays. Methods, 25, 430-442). Measurement of T/R ratios bymultiplex QPCR cuts in half the number of separate PCR reactions thatmust be run. Furthermore, since both T and R signals are collectedwithin each reaction vessel, variation in the amount of a given DNAsample that is pipetted for replicate reactions no longer generatesvariation in the T/R ratios, as it does when T and R are measured inseparate wells in monoplex QPCR.

The main disadvantage of multicolor multiplex QPCR is the relativelyhigh cost of the fluorescent probes, and the high cost of thespecialized QPCR machines that are equipped to read two or morefluorescent colors. In traditional approaches to multiplex PCR (whetheror not the PCR is quantitative), it is also sometimes excessivelytime-consuming to identify primer sets and primer concentrations thatprevent the earlier amplification of a higher copy number template byone primer pair from inhibiting the later amplification of a different,lower copy number template by a second primer pair

SUMMARY OF THE INVENTION

The present invention provides methods of determining the copy number oftwo or more target nucleic acid sequences in a single reaction with asingle detection label. Also disclosed are methods for determining thecopy number of telomeric sequences. This data may be used forassociating the measured telomere length with a mortality risk orlikelihood of disease occurrence that corresponds to a telomere lengthobserved in a population.

Disclosed herein are methods and compositions for determining the copynumber of a first target nucleic acid as compared to the copy number ofa second target nucleic acid by a monochrome multiplex quantitative PCR(MMQPCR) in a single well, a homogenous system, with a single detectionlabel.

Additional advantages of the disclosed methods and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or can be learned by practice of thedisclosed methods and compositions. The advantages of the disclosedmethods and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed methods and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 shows in Cycle 1 the telg primer hybridizes to native telomeresequences and primes DNA synthesis. The telc primer hybridizes nativetelomere sequences but cannot prime DNA synthesis, due to its 3′terminal mismatch. When hybridized to each other as shown, and in otherconfigurations not shown, telg and telc have multiple mismatches,including at their 3′ terminal bases, so primer dimer formation isinhibited. The 3′ ends of telg and telc can align as a perfectlycomplementary three by overlap, it is not stable enough to allowefficient primer dimer formation. In Cycle 2, telc can hybridize alongtelg primer extension products that were synthesized in Cycle 1, but canonly prime DNA synthesis when hybridized in the configuration shown,since other configurations produce a mismatch at telc's 3′ terminalbase. In the telg extension product, the overbar marks the sequence ofthe telg primer itself, and the italicized bases mark sequence newlysynthesized in Cycle 1 of the PCR. The non-templated capitalizedsequences at the 5′ ends of the primers prevent the 3′ ends of thetelomere PCR product from priming DNA synthesis in the middle of othercopies of the telomere PCR product.

FIG. 2 shows the melting curves following 25 cycles of amplification(thermal profile given in Materials and Methods section) of 150 ng ofhuman genomic DNA with telomere primers only (circles), albumin primersonly (“x”s), or both primer sets (triangles). No template controlmelting curves are in black with no symbols. After the final 88° C.incubation, reactions were cooled to 72° C., and signal was acquiredfrom 72° C. to 95° C., in 0.5° C. steps, with a 30 second dwell periodper step. There is approximately an 11° C. difference in the meltingtemperatures of the telomere and albumin amplicons.

FIG. 3 shows Monochrome Multiplex Quantitative PCR (MMQPCR) of 20 ng ofeach of three reference human DNA samples previously shown to have longtelomeres (circles), middle-length telomeres (“x”s), or short telomeres(triangles). No template control amplification curves are shown withblack squares. Top panel: semi-log plot; bottom panel: linear plot.

FIG. 4 shows standard curves used to determine relative T/S ratios. Fiveconcentrations of a standard human genomic DNA sample spanning an81-fold range were prepared by 3-fold serial dilutions (150 ng, 50 ng,16.7 ng, 5.55 ng, and 1.85 ng per well), and aliquoted in duplicate to a96-well PCR plate. Both target and reference fluorescent signals werecollected from each reaction well. Circles represent data for the singlecopy gene albumin, acquired at 88° C.; triangles, data for telomererepeats, acquired at 74° C. The same Standard DNA was used to set upstandard curve reactions in every plate in the study.

FIG. 5 shows correlation of relative T/S ratios measured by MonochromeMultiplex Quantitative PCR with albumin as the single copy gene, andmean Terminal Restriction Fragment (TRF) lengths determined by Southernblot analysis, in whole blood DNA samples from 95 individuals. Each T/Svalue is the average of triplicate measurements; each mean TRF length isthe average of duplicate measurements. The linear regression equationand correlation coefficient were determined using Microsoft Excel.

FIG. 6 shows reproducibility of relative T/S ratios in independent runsof the MMQPCR assay. The same 95 DNA samples assayed in FIG. 4 wereassayed again the next day, taking care that the specific MyiQ PCRmachine and reaction well positions occupied by each DNA sample weredifferent from the previous day. The linear regression equation andcorrelation coefficient were determined using Microsoft Excel.

FIG. 7 shows the correlation between T/S ratios obtained with albumin asthe single copy gene vs. beta-globin as the single copy gene. RelativeT/S ratios were measured in the same 95 DNA samples, in triplicate, intwo separate runs, substituting the beta-globin primers for the albuminprimers. For each sample, the average T/S from the two separate runswith albumin as the single copy gene (x axis) is plotted against theaverage T/S from the two runs with beta-globin as the single copy gene(y axis). The linear regression equation and correlation coefficientwere determined using Microsoft Excel.

DETAILED DESCRIPTION

The present invention comprises methods and systems directed atdetermining the copy number of one or more target nucleic acids. Thedisclosed method and compositions can be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

The disclosed compositions and methods can be used for real-timedetection of target nucleic acids. Real-time detection is detection cantake place during or immediately after the amplification reaction oroperation. Generally, such detection can be accomplished by detectingamplification product at one or more discrete times duringamplification, continuously during all or one or more portions of theamplification, or a combination of discrete times and continuousdetection. Real-time detection can be aided by the use of labels ormoieties that embody or produce a detectable signal that can be detectedwithout disrupting the amplification reaction or operation. Fluorescentlabels are an example of useful labels for real-time detection. Aparticularly useful means of obtaining real-time detection is the use ofdetection labels in the amplification operation. With suitably designeddetection labels, detection signals, including fluorescent signals, canbe generated as amplification proceeds. In most such cases, thedetection signals will be in proportion to the amount of amplificationproduct and/or amount of target sequence or target molecule.

Disclosed herein are methods for determining the copy number of a firsttarget nucleic acid and a second target nucleic acid. A target nucleicacid can be obtained from a sample or artificially generated asdescribed elsewhere herein.

All patents, patent applications and publications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretiesinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

It is to be understood that this invention is not limited to specificsynthetic methods, or to specific recombinant biotechnology methodsunless otherwise specified, or to particular reagents unless otherwisespecified, to specific pharmaceutical carriers, or to particularpharmaceutical formulations or administration regimens, as such may, ofcourse, vary.

DEFINITIONS AND NOMENCLATURE

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” can include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a compound”includes mixtures of compounds, reference to “a pharmaceutical carrier”includes mixtures of two or more such carriers, and the like. Referenceto “a component” can include a single or multiple components or amixtures of components unless the context clearly dictates otherwise.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 20%. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; or a solution containing one or moremolecules derived from a cell or cellular material (e.g. a polypeptideor nucleic acid), which is assayed as described herein. A sample mayalso be any body fluid or excretion (for example, but not limited to,blood, urine, stool, saliva, tears, bile) that contains cells or cellcomponents.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combinationthereof.

By “specifically binds” is meant that the composition recognizes andphysically interacts with its cognate target. For example, a primer canspecifically bind to its target nucleic acid. For example, a primer ofthe first primer set can specifically bind to the first target nucleicacid sequence and does not significantly recognize and interact withother targets or target nucleic acid sequences.

By “probe,” “primer,” or oligonucleotide is meant a single-stranded DNAor RNA molecule of defined sequence that can base-pair to a second DNAor RNA molecule that contains a complementary sequence (the “target”).The stability of the resulting hybrid depends upon the extent of thebase-pairing that occurs. The extent of base-pairing is affected byparameters such as the degree of complementarity between the probe andtarget molecules and the degree of stringency of the hybridizationconditions. The degree of hybridization stringency is affected byparameters such as temperature, salt concentration, and theconcentration of organic molecules such as formamide, and is determinedby methods known to one skilled in the art. Probes or primers specificfor target nucleic acids (for example, genes and/or mRNAs) have at least80%-90% sequence complementarity, at least 91%-95% sequencecomplementarity, at least 96%-99% sequence complementarity, or at least100% sequence complementarity to the region of the target to which theyhybridize. Probes, primers, and oligonucleotides may bedetectably-labeled, either radioactively, or non-radioactively, bymethods well-known to those skilled in the art. Probes, primers, andoligonucleotides are used for methods involving nucleic acidhybridization, such as: the described monochrome multiplex quantitativePCR (MMQPCR) described herein as well as nucleic acid sequencing,reverse transcription and/or nucleic acid amplification by thepolymerase chain reaction, single stranded conformational polymorphism(SSCP) analysis, restriction fragment polymorphism (RFLP) analysis,Southern hybridization, Northern hybridization, in situ hybridization,electrophoretic mobility shift assay (EMSA).

By “primer set” is meant to mean at least two primers that each containa complementary sequence to an opposite strand of the same targetsequence. In a primer set, at least one of the two primers must be a“forward primer” at least one of the two primers must be a “reverseprimer”. A “forward primer” is a primer that is complementary to a sensestrand of a target nucleic acid, wherein a “reverse primer” is a primerthat is complementary to the complement of the sense strand of thetarget nucleic acid (also referred to as the anti-sense strand of thetarget nucleic acid). A primer set can be a pair of primers capable ofbeing used in a PCR reaction.

By “amplicon” is meant to mean pieces of DNA formed as the products ofnatural or artificial amplification events. For example, they can beformed via the methods described herein, polymerase chain reactions(PCR) or ligase chain reactions (LCR), as well as by natural geneduplication.

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a target nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant conditions that allowhybridization comparable with that resulting from the use of a DNA probeof at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well-known by thoseskilled in the art of molecular biology. (See, for example, F. Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1998).

Materials

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. Thus, if a class of molecules A, B, and C are disclosed as wellas a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited, each is individually and collectively contemplated. Thus, isthis example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D,C-E, and C-F are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. Thus, for example, thesub-group of A-E, B-F, and C-E are specifically contemplated and shouldbe considered disclosed from disclosure of A, B, and C; D, E, and F; andthe example combination A-D. This concept applies to all aspects of thisdisclosure including, but not limited to, steps in methods of making andusing the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods, and that each suchcombination is specifically contemplated and should be considereddisclosed.

A. Target Samples

Target samples can be derived from any source that has, or is suspectedof having, target molecules. Target samples can contain, for example, atarget molecule(s) such as nucleic acids. A target sample can be thesource of target nucleic acids. A target sample can include naturaltarget nucleic acids, chemically synthesized target nucleic acids, orboth. A target sample can be, for example, a sample from one or morecells, tissue, or bodily fluids such as blood, urine, semen, lymphaticfluid, cerebrospinal fluid, or amniotic fluid, or other biologicalsamples, such as tissue culture cells, buccal swabs, mouthwash, stool,tissues slices, biopsy aspiration, and archeological samples such asbone or mummified tissue. Types of useful target samples include bloodsamples, urine samples, semen samples, lymphatic fluid samples,cerebrospinal fluid samples, amniotic fluid samples, biopsy samples,needle aspiration biopsy samples, cancer samples, tumor samples, tissuesamples, cell samples, cell lysate samples, crude cell lysate samples,forensic samples, archeological samples, infection samples, nosocomialinfection samples, production samples, drug preparation samples,biological molecule production samples, protein preparation samples,lipid preparation samples, and/or carbohydrate preparation samples.

1. Target Nucleic Acids

Nucleic acid samples can be derived from any source that has, or issuspected of having, target nucleic acids. A nucleic acid sample is thesource of nucleic acid molecules and nucleic acid sequences such astarget nucleic acids. The nucleic acid sample can contain RNA or DNA orboth. The target nucleic acid can also be cDNA. In addition, mRNA can bereverse transcribed to form cDNA which can then serve as a targetnucleic acid for use in the methods described herein.

A “target nucleic acid” or “target sequence” is meant a nucleic acidsequence on a double or single stranded nucleic acid. By “nucleic acid”or “oligonucleotide” or grammatical equivalents herein is meant at leasttwo nucleotides covalently linked together. A nucleic acid of thepresent invention will generally contain phosphodiester bonds, althoughin some cases, nucleic acid analogs are included that may have alternatebackbones, comprising, for example, phosphoramide (Beaucage, S. L. etal., Tetrahedron 49: 1925-63 (1993), and references therein; Letsinger,R. L. et al., J. Org. Chem. 35: 3800-03 (1970); Sprinzl, M. et al., Eur.J. Biochem. 81: 579-89 (1977); Letsinger, R. L. et al., Nucleic AcidsRes. 14:3487-99 (1986); Sawai et al, Chem. Lett. 805 (1984); Letsinger,R. L. et al., J. Am. Chem. Soc. 110: 4470 (1988); and Pauwels et al.,Chemica Scripta 26:141-49 (1986)), phosphorothioate (Mag, M. et al.,Nucleic Acids Res. 19:1437-41 (1991); and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989)),O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press, 1991), andpeptide nucleic acid backbones and linkages (Egholm, M., Am. Chem. Soc.114:1895-97 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992);Egholm, M., Nature 365: 566-68 (1993); Carlsson, C. et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analognucleic acids include those with positive backbones (Dempcy, R. O. etal., Proc. Natl. Acad. Sci. USA 92:6097-101 (1995)); non-ionic backbones(U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger, R. L. et al., J. Am. Chem. Soc. 110: 4470 (1988);Letsinger, R. L. et al., Nucleoside & Nucleotide 13: 1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4: 395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994)) and non-ribose backbones, including thosedescribed in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and7, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containingone or more carbocyclic sugars are also included within the definitionof nucleic acids (see Jenkins et al., Chem. Soc. Rev. 169-176 (1995));all references are hereby expressly incorporated by reference.

Any nucleic acid sequence sought to be measured, identified, detected orwhose copy number is sought to be determined can serve as a targetnucleic acid sequence. In the methods described herein, there can bemore than one target nucleic acid sequence. In the event that two targetnucleic acid sequences are present, they will be referred to as a firstand second target nucleic acid sequence, respectfully. In the event thatthree target nucleic acid sequences are present, they will be referredto as a first, a second and a third target nucleic acid sequence,respectfully and so on. The target nucleic acids described in themethods herein can have the same, similar or different copy numbers. Forexample, the first target nucleic acid is a nucleic acid sequence ofmultiple copy numbers and the second target nucleic acid is a singlecopy gene. For example, the first target nucleic acid can be telomericrepeat sequences, mtDNA, rDNA or Alu repeat DNA. For example, the firsttarget nucleic acid can be cDNA reverse-transcribed from a high copynumber mRNA, and the second target nucleic acid can be cDNAreverse-transcribed from a low copy number mRNA.

Single copy genes are genes that have a single copy per haploid genome.Single copy genes therefore have two copies per cell. Single copy genesinclude, but are not limited to, the albumin gene or the beta-globingene.

Telomeres are specialized structures found at the ends of linearchromosomes of eukaryotes. Telomeres are generally composed of shorttandem repeats, with a repeat sequence unit specified by the telomeraseenzyme particular to that organism. Telomere repeat sequences are knownfor a variety of organisms. For vertebrates, plants, certain types ofmolds, and some protozoans, the sequences are perfect repeats. Forexample, the human repeat sequence unit is (TTAGGG)n. (SEQ ID NO:1) Inother organisms, the repeats sequences are irregular, such as those ofSacharomyces cerevisiae where the sequence is variable G1-3 T/C1-3A. Insome eukaryotic organisms, telomeres lack the short tandem sequencerepeats but have sequence elements that function as telomeres. Forexample, in the fruit fly Drosophila melanogaster, the telomere is acomposite of retrotransposon elements HeT-A and TART while in themosquito Anopheles gambiae the telomeres are arrays of complex sequencetandem repeats. For the purposes of the present invention, telomeres ofdifferent structures are encompassed within the scope of the presentinvention.

In addition to the repeat sequences, the 3′ end of some telomerescontains a single stranded region, which for humans is located on the Grich strand. The single strand is composed of (TTAGGG)_(n). (SEQ IDNO:1) repeats, with n being generally about 9-35, although it can beless or more. As used herein, the length of the 3′ single strandedregion can also be correlated with mortality risk.

Typically, the DNA replicative machinery acts in the 5′ to 3′ direction,and synthesis of the lagging strand occurs discontinuously by use ofshort RNA primers that are degraded following strand synthesis. Sincesequences at the 3′ end of a linear DNA are not available to completesynthesis of the region previously occupied by the RNA primer, theterminal 3′ region of the linear chromosome is not replicated. This “endreplication problem” is solved by the action of telomerase, a telomerespecific ribonucleoprotein reverse transcriptase. The telomerase enzymehas an integral RNA component that acts as a template for extending the3′ end of the telomere. Repeated extensions by telomerase activityresults in the generation of telomere repeats copied from thetelomerase-bound RNA template. Following elongation by telomerase,lagging strand synthesis by DNA polymerase completes formation of thedouble stranded telomeric structure.

In normal human somatic cells, telomerase is not expressed or expressedat low levels. Consequently, telomeres shorten by 50-200 bp with eachcell division until the cells reach replicative senescence, at whichpoint the cells lose the capacity to proliferate. This limited capacityof cells to replicate is generally referred to as the Hayflick limit,and may provide cells with a counting mechanism, i.e., a mitotic clock,to count cell divisions and regulate cellular development.Correspondingly, activation of telomerase in cells lacking telomeraseactivity, for example by expressing telomerase from a constituteretroviral promoter or activation of endogenous polymerase, allows thecells to maintain proliferative capacity and leads to immortalization ofthe cell.

Interestingly, these immortalized cells have short stable telomereswhile the shortest telomeres become extended. This phenomena suggeststhat telomerase enzyme protects short telomeres from further shorteningwhile extending those that have fallen below a certain threshold length.Thus, presence of telomerase activity does not appear to be necessarywhen telomeres are a certain length, but becomes critical to maintenanceof telomere integrity when the length falls below a critical limit.

It is well established that the length and integrity of telomeres isimportant for proper segregation of chromosomes and cell growth. Forexample, development of many types of cancers correlates with activationof telomere maintenance while cell senescence correlates with loss oftelomere integrity. Shortening of telomere induced by inhibitingtelomerase activity can lead to proliferative senescence and cellapoptosis (Zhang, X. et al., Genes Dev. 2388-99 (1999)). Moreover,genetic knockouts of telomerase RNA in mice results in animals withdevelopmental defects, age related pathologies, and increased cancersusceptibility (Rudolph, K. L. et al., Cell 96: 701-12 (1999); Herrera,E. et al., EMBO J. 18: 2950-60 (1999)). Similarly, in the autosomaldominant disorder of dyskeratosis congenital (DKC), which arises from amutation in the gene encoding the RNA component of telomerase, afflictedpatients display accelerated telomere shortening and die at a median ageof 16 years (maximum approximately 50 years), usually from severeinfections secondary to bone marrow failure. Clinical features of DKCpatients, further suggestive of accelerated aging, include prematuregraying and loss of hair; skin dyspigmentation; poor wound healing; highrisk of severe infections; and an increased incidence of malignancies,osteoporosis, and pulmonary fibrosis. In addition, the shortest averagetelomere lengths measured in blood DNA from normal elderly individualsoverlap with the highest average telomere lengths measured in blood fromDKC patients.

In view of the role telomeres play in cell growth and cell senescence,it is desirable to have methods of predicting the occurrence of agerelated diseases and mortality risk based on length of telomeres. Themethods described herein, including the MMQPCR methods, will provide abasis for identifying individuals with increased risk of developingparticular age-associated diseases, such as cancer and hypertension,such that early medical intervention can be administered to individualsin high risk groups.

In the methods described herein, the copy number of a telomere may bedetermined for a single chromosome in a cell. In an aspect, the averagecopy number of a telomere or mean telomere copy number is measured for asingle cell. In another embodiment, the average copy number of atelomere or mean telomere copy number is measured for a population ofcells. A change in telomere copy number is an increase or decrease intelomere copy number, in particular an increase or decrease in theaverage telomere copy number. The change may be relative to a particulartime point, i.e., telomere copy number of an organism at time t1 ascompared to telomere length at some later time t2. A change ordifference in telomere copy number may also be compared as against theaverage or mean telomere copy number of a particular cell population ororganism population. In some aspects, a change or difference in telomerecopy number may also be compared as against the average or mean telomerecopy number of a population not suffering from a disease condition. Incertain embodiments, change in telomere copy number is measured againsta population existing at different time periods.

Although, telomere copy number may be determined for all eukaryotes, ina one aspect, telomere copy numbers are determined for vertebrates,including without limitation, amphibians, birds, and mammals, forexample rodents, ungulates, and primates, particularly humans. Telomerecopy numbers can also be determined for organisms in which longevity isa desirable trait or where longevity and susceptibility to disease arecorrelated. In another aspect, the telomeres may be measured for clonedorganisms in order to assess the mortality risk or diseasesusceptibility associated with altered telomere integrity in theseorganisms.

Telomeric nucleic acid sequences, such as those described above canserve as a target sequence. Telomeric nucleic acid sequences, or anyother target nucleic acid, may be any length, with the understandingthat longer sequences are more specific. In some embodiments, it may bedesirable to fragment or cleave the sample nucleic acid into fragmentsof 100-10,000 base pairs. In one aspect, fragments of roughly 500basepairs can be used. Fragmentation or cleavage may be done in anynumber of ways well known to those skilled in the art, includingmechanical, chemical, and enzymatic methods. Thus, the nucleic acids maybe subjected to sonication, French press, shearing, or treated withnucleases (e.g., DNase, restriction enzymes, RNase etc.), or chemicalcleavage agents (e.g., acid/piperidine, hydrazine/piperidine, iron-EDTAcomplexes, 1,10-phenanthroline-copper complexes, etc.).

2. Polymerases

In the methods described herein, an amplification enzyme is required.For example, following contacting the primers to the target nucleicacids, the reaction can be treated with an amplification enzyme.Amplification enzymes are generally polymerases, such as DNApolymerases. A variety of suitable polymerases are well known in theart, including, but not limited to, Taq DNA polymerase, KlenTaq, Tflpolymerase, DynaZyme, etc. Generally, all polymerases are applicable tothe present invention. In one aspect, polymerases are thermostablepolymerases lacking 3′ to 5′ exonuclease activity, or polymerasesengineered to have reduced or non-functional 3′ to 5′ exonucleaseactivities (e.g., Pfu(exo-), Vent(exo-), Pyra(exo-), etc.), since use ofpolymerases with strong 3′ to 5′ exonuclease activity tends to removethe mismatched 3′ terminal nucleotides that are needed in someapplications to prevent or delay primer dimer amplifications, and inother applications to carry out allele-specific amplifications. Alsoapplicable are mixtures of polymerases used to optimally extendhybridized primers. In another aspect, polymerase enzymes useful for thepresent invention are formulated to become active only at temperaturessuitable for amplification.

Presence of polymerase inhibiting antibodies, which become inactivatedat amplification temperatures, or sequestering the enzymes in a formrendering it unavailable until amplification temperatures are reached,are all suitable. These polymerase formulations allow mixing allcomponents in a single reaction vessel while preventing priming ofnon-target nucleic acid sequences.

In addition, those skilled in the art will appreciate that variousagents may be added to the reaction to increase processivity of thepolymerase, stabilize the polymerase from inactivation, decreasenon-specific hybridization of the primers, or increase efficiency ofreplication. Such additives include, but are not limited to, dimethylsulfoxide, formamide, acetamide, glycerol, polyethylene glycol, orproteinacious agents such as E. coli. single stranded DNA bindingprotein, T4 gene 32 protein, bovine serum albumin, gelatin, etc. Inanother aspect, the person skilled in the art can use various nucleotideanalogs for amplification of particular types of sequences, for exampleGC rich or repeating sequences. These analogs include, among others,c7-dGTP, hydroxymethyl-dUTP, dITP, 7-deaza-dGTP, etc.

3. Primers

By “primer”, “primer nucleic acid”, “oligonucleotide primer”,“oligonucleotide probe” or grammatical equivalents as used herein ismeant a nucleic acid that will hybridize to some portion of a targetnucleic acid. The primers or probes of the present invention aredesigned to be substantially complementary to a target sequence suchthat hybridization of the target sequence and the primers of the presentinvention occurs.

In some aspects, a primer can be designed to block the primer frompriming extension of the target nucleic acid in all but oneconfiguration. For example, one of the primers in a primer set can bedesigned to block the primer from priming the extension of the targetnucleic acid by creating a mismatched base at the 3′ end of the primer.By designing and utilizing such a primer, the primer is still able tohybridize to its complementary sequence; however, it will only prime DNAsynthesis is a single confirmation, thus giving predictability to theamplicon size and therefore predictability to the Tm of the amplicon.

For example, disclosed herein are primers and primer sets, wherein oneprimer of the first primer set comprises at least one nucleotideadjacent to the 3′ end of the primer, wherein said nucleotide ismismatched against, not complementary to, the target nucleic acid, butcomplementary to the 3′ terminal nucleotide of the other primer in theprimer set.

Also disclosed herein are primers and primer sets, wherein one primer ofa primer set comprises at least one nucleotide adjacent to the 3′ end ofthe primer, wherein said nucleotide is mismatched against, notcomplementary to, the target nucleic acid, but complementary to the 3′terminal nucleotide of the other primer in the primer set, wherein theextension product of the mismatch-containing primer of the primer setcan be hybridized by the other primer in the primer set, allowing saidother primer to prime DNA synthesis along said extension product.

To ensure that a blocked primer will only prime in a single, specificconfiguration, a primer set including the blocked primer can be designedsuch that the primers of the primer set overlap with perfectcomplementarity over the region of the mismatched base present in theblocked primer. Such a design can be performed so as to prevent primerdimer formation and to minimize the ability of the two primers to primeeach other. Such a design can be utilized when the target nucleic acidsequence is a sequence comprising multiple repeats such as the repeatsfound in a telomere (telomeric sequence). An example of such a method isdescribed elsewhere herein, including the Examples below.

As described herein, the primers for direct amplification of telomererepeats can comprise a first primer which hybridizes to a first singlestrand of the target nucleic acid and a second primer which hybridizesto a second single strand of the target nucleic acid, where the firstand second strands are substantially complementary. The primers arecapable of primer extension by polymerase when hybridized to theirrespective strands. That is, the primers hybridized to the targetnucleic acid have their 3′ terminal nucleotide residues complementary tothe nucleotide residue on the target nucleic acid such that the primersare extendable by polymerase. Selected primers are complementary torepetitive units of the repetitive region. For example, at least onenucleotide residue of at least one of the primers can be altered toproduce mismatches with a nucleotide residue of at least one repetitiveunit to which the primer hybridizes, wherein the altered nucleotideresidue also produces a mismatch with the 3′ terminal nucleotide residueof the other primer when the primers hybridize to each other. Theinclusion of a mismatch prevents or limits primer extension andprimer-primer hybrids.

A primer set for direct amplification of telomere repeats can comprise aprimer set wherein at least one nucleotide residue of the first primeris altered to produce a mismatch between the altered residue and anucleotide residue of at least one repetitive unit of the first strandto which the primer hybridizes, wherein the altered nucleotide residuealso produces a mismatch with the 3′ terminal nucleotide residue of thesecond primer when the first and second primers hybridize to each other.The altered nucleotide residue can be one or more nucleotide residuesfrom the 3′ terminal nucleotide to allow efficient extension bypolymerase when the altered primer hybridizes to target nucleic acids.For example, the altered nucleotide residue can be at least 1 nucleotideresidue, at least 2 nucleotide residues, or at least 3 nucleotideresidues from the 3′ terminal nucleotide to allow efficient extension bypolymerase when the altered primer hybridizes to target nucleic acids.

As discussed elsewhere herein, the primers of the primer sets can bedesigned to have similar Tms to limit generation of undesirableamplification products and to permit amplification and detection ofseveral target nucleic acids in a single reaction volume. In addition,since the telomeres of various organisms have differing repetitive unitsequences, amplifying telomeres of a specific organism will employprimers specific to the repetitive unit of each different organism.Human telomeric sequences are used herein to illustrate practice of thepresent invention for direct amplification and quantitation of tandemlyrepeated nucleic acid sequences, but the invention is not limited to thedisclosed specific embodiment.

Also disclosed are primers to increase the melting temperature (Tm) ofthe resultant amplicon above that of the other amplicon of the methodsdescribed herein. These primers can be referred to as primers comprisinga “GC-clamp”. “GC-clamps” typically refers to the presence of G or Cbases within the last five bases from the 3′ end of primers that helpspromote specific binding at the 3′ end due to the stronger bonding of Gand C bases. Typically, more than 3 G's or C's should be avoided in thelast 5 bases at the 3′ end of the primer. However, in the methodsdescribed herein primers comprising a “GC-clamp” are primers thatcomprise a 5′ tag sequence (GC-clamp) that confers a higher meltingtemperature on the resulting PCR product (amplicon) than the meltingtemperature without the GC-clamp. The 5′ tag sequence of primerscomprising a “GC-clamp” comprise a GC-clamp on the 5′ end of the primersequence that is not complementary to any part of the target nucleicacid sequence. A “GC-clamp” is a series of G and C nucleotides that canbe linked to the 5′ end of a primer in order to increase the meltingtemperature of the amplicon. A GC-clamp can be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more nucleotides long. A GC-clamp can also be referredto a GC-rich region or GC-rich tag.

GC-clamps can be used in the methods described herein to increase the Tmof one of the amplicons. By increasing the Tm of the amplicon, afluorescent signal can be acquired at a temperature high enough tocompletely melt the other amplicon, thus allowing for the acquisition ofa fluorescent signal for two or more different amplicons at two or moredifferent temperatures. GC-clamped primers can be designed for use inthe same amplification reaction such that the GC-clamps on differentprimers are different from one another so as to prevent hairpinformation or primer dimers that could result in a cessation of theamplification reaction.

Since primers hybridized to target nucleic acids must be capable ofprimer extension, alterations of the first and second primers must be onnon-complementary nucleotides of the repetitive unit. Thus, in oneaspect, when both the first and second primers comprise alteredresidues, the alterations are at nucleotide positions adjacent to therepetitive unit. In another aspect, the alterations are situated onnucleotide positions non-adjacent to the repetitive unit. In general,mismatches at adjacent nucleotide positions provide for the most numberof base paired or complementary residues between the altered nucleotideand the 3′ terminal nucleotide, which may be important for efficientlyamplifying short repetitive sequences (i.e., 3-6 basepairs).

Complementarity of the primers to the target nucleic acid need not beperfect. Thus, by “complementary” or “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions.Deviations from perfect complementary are permissible so long asdeviations are not sufficient to completely preclude hybridization.However, if the number of alterations or mutations is sufficient suchthat no hybridization can occur under the least stringent ofhybridization conditions, as defined below, the sequence is not acomplementary target sequence.

Although primers are generally single stranded, the nucleic acids asdescribed herein may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, RNA, or hybrid, wherethe nucleic acid contains any combination of deoxyribo- andribonucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, xanthine hypoxanthine, isocytosine,isoguanine, inosine, etc. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thus,for example, the individual units of a peptide nucleic acid, eachcontaining a base, are referred herein as a nucleotide.

The size of the primer nucleic acid may vary, as will be appreciated bythose in the art, in general varying from 5 to 500 nucleotides inlength. For example, with primers of between 10 and 100 nucleotides,between 12 and 75 nucleotides, and from 15 to 50 nucleotides can beused, depending on the use, required specificity, and the amplificationtechnique.

For any primer pair, the ability of the primers to hybridize to eachother may be examined by aligning the sequence of the first primer tothe second primer. The stability of the hybrids, especially the thermalmelting temperature (Tm), may be determined by the methods describedbelow and by methods well known in the art. These include, but are notlimited to, nearest-neighbor thermodynamic calculations (Breslauer, T.et al., Proc. Natl. Acad. Sci. USA 83:8893-97 (1986); Wetmur, J. G.,Crit. Rev. Biochem. Mol. Biol. 26:227-59 (1991); Rychlik, W. et al., J.NIH Res. 6:78 (1994)), Wallace Rule estimations (Suggs, S. V. et al “Useof Synthetic oligodeoxyribonucleotides for the isolation of specificcloned DNA sequences,” Developmental biology using purified genes, D. B.Brown, ed., pp 683-693, Academic Press, New York (1981), and Tmestimations based on Bolton and McCarthy (see Baldino, F. J. et al.,Methods Enzymol. 168: 761-77 (1989); Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Chapter 10, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (2001)). All references are herebyexpressly incorporated by reference. The effect of various parameters,including, but not limited to, ionic strength, probe length, G/Ccontent, and mismatches are taken into consideration when assessinghybrid stability. Consideration of these factors are well known to thoseskilled in the art (see, e.g., Sambrook, J., supra).

The primers that can be used in the methods described herein can be usedto amplify various target nucleic acids. A single primer set, forexample a primer pair, may be used to amplify a single target nucleicacid. In another embodiment, multiple primer sets may be used to amplifya plurality of target nucleic acids. Amplifications may be conductedseparately for each unique primer set, or in a single reaction vesselusing combinations of primer sets, generally known in the art asmultiplexing. When multiple primer sets are used in a single reaction,primers are designed to limit formation of undesirable products andlimit interference between primers of each primer set.

The general PCR amplification reactions can be carried out according toprocedures well known in the art, as discussed above (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202). The time and temperature of theprimer extension step will depend on the polymerase, length of targetnucleic acid being amplified, and primer sequence employed for theamplification. The number of reiterative steps required to sufficientlyamplify the target nucleic acid will depend on the efficiency ofamplification for each cycle and the starting copy number of the targetnucleic acid. As is well known in the art, these parameters can beadjusted by the skilled artisan to effectuate a desired level ofamplification. Those skilled in the art will understand that the presentinvention is not limited by variations in times, temperatures, bufferconditions, and the amplification cycles applied in the amplificationprocess.

In hybridizing the primers to the target nucleic acids and in thedisclosed amplification reactions, the assays are generally done understringency conditions that allow formation of the hybrids in thepresence of target nucleic acid. Those skilled in the art can alter theparameters of temperature, salt concentration, pH, organic solvent,chaotropic agents, or other variables to control the stringency ofhybridization and also minimize hybridization of primers to non-specifictargets (i.e., by use of “hot start” PCR or “touchdown” PCR).

4. Detection Labels

To aid in determining the copy number of target nucleic acids using thedisclosed compositions and methods, detection labels can be directlyincorporated into amplified nucleic acids or can be coupled to detectionmolecules. As used herein, a detection label is any molecule that can beassociated with amplified nucleic acid, directly or indirectly, andwhich results in a measurable, detectable signal, either directly orindirectly. In the methods described herein, a single detection labelcan be used. By a “single detection label” is meant to mean a singletype of detection label. For example, a single detection label can beany detection label as described herein, however only one type ofdetection label can be used in each homogenous system. For example, asingle detection label can be SYBR Green I (Invitrogen), fluoresceinisothiocyanate (FITC), 5,6-carboxymethyl fluorescein, or Texas red butnot a combination of all of these. Thus, SYBR Green I (Invitrogen),fluorescein isothiocyanate (FITC), 5,6-carboxymethyl fluorescein, orTexas red are each single detection labels, for example, SYBR Green I(Invitrogen) is a single detection label, fluorescein isothiocyanate(FITC) is a single detection label, 5,6-carboxymethyl fluorescein is asingle detection label, and Texas red is a single detection label.

Another example is provided in the Examples below where SYBR Green isthe “single detection label” used to determine the copy number of twodifferent targets. In addition, a single detection label can also bereferred to as a single, monochrome, detection label. A “single,monochrome detection label” is a single detection label that has onlyone color. For example, a single, monochrome detection label can be adetection label that emits a single color that can be detected.

Many such labels for incorporation into nucleic acids or coupling tonucleic acid probes are known to those of skill in the art. Examples ofdetection labels suitable for use in the disclosed method areradioactive isotopes, phosphorescent molecules, enzymes, antibodies, andligands as well as fluorescent molecules including fluorescent dyes andfluorescent labels. Fluorescent labels are useful for real-timedetection of amplification.

For example, the methods described herein can use fluorescent dyes thatpreferentially bind to double stranded nucleic acid amplificationproducts during the PCR reaction, thereby providing continuousmonitoring of product synthesis (see Higuchi, R. et al., Biotechnology11: 1026-1030 (1993); Morrison, T. B. et al., Biotechniques 24: 954-962(1998)).

Examples of suitable fluorescent labels include, but are not limited to,SYBR Green I (Invitrogen), fluorescein isothiocyanate (FITC),5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY®,Cascade Blue®, Oregon Green®, pyrene, lissamine, xanthenes, acridines,oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such asquantum Dye™, fluorescent energy transfer dyes, such as thiazoleorange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7. Examples of other specific fluorescent labels include3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT),Acid Fuchsin, Alizarin Complexon, Alizarin Red, Allophycocyanin,Aminocoumarin, Anthroyl Stearate, Astrazon Brilliant Red 4G, AstrazonOrange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine,Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF,Berberine Sulphate, Bisbenzamide, Blancophor FFG Solution, BlancophorSV, Bodipy F1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green,Calcofluor RW Solution, Calcofluor White, Calcophor White ABT Solution,Calcophor White Standard Solution, Carbostyryl, Cascade Yellow,Catecholamine, Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.18, CY5.1 8, CY7, Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid),Dansa (Diamino Naphtyl Sulphonic Acid), Dansyl NH—CH3, Diamino PhenylOxydiazole (DAO), Dimethylamino-5-Sulphonic acid, DipyrrometheneboronDifluoride, Diphenyl Brilliant Flavine 7GFF, Dopamine, Erythrosin ITC,Euchrysin, FIF (Formaldehyde Induced Fluorescence), Flazo Orange, Fluo3, Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl BrilliantYellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid,Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, LeucophorPAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, MaxilonBrilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (MethylGreen Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole,Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan BrilliantFlavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), PhorwiteAR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black,Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron BrilliantRed 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange,Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonicacid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine GExtra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN,Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue,Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC.Fluorescentlabels can be obtained from a variety of commercial sources, includingInvitrogen, Carlsbad, Calif.; Amersham Pharmacia Biotech, Piscataway,N.J.; Molecular Probes, Eugene, Oreg.; and Research Organics, Cleveland,Ohio.

5. Instruments

Instrumentation suitable for use with the disclosed methods andcompositions include but are not limited to ABI Prism 7700, AppliedBiosystems Division, Perkin Elmer, Fosters City, Calif., USA;LightCycler™, Roche Molecular Biochemicals, Indianapolis, Ind., USA.

Various algorithms can be used to calculate the copy number of thetarget nucleic acids in the samples as described herein (see ABI Prism7700 Software Version 1.7; Lightcycler™ Software Version 3; incorporatedby reference). Determining copy number may involve use of standardsamples with known copy number of the target nucleic acid and generationof standard curves from the logarithms of the standards and the cycle ofthreshold (Ct). In general, Ct is the PCR cycle or fractional PCR cyclewhere the fluorescence generated by the amplification product is severaldeviations above the baseline fluorescence (Higuchi, R. et al., supra).MMQPCR provides a linearity of about 7 to 8 orders of magnitude, whichallows measurement of copy number of target nucleic acids over a widedynamic range. The absolute number of target nucleic acid copies can bederived from comparing the Ct values of the standard curve and thesamples.

The copy number of target nucleic acids may also be determined bycomparative MMQPCR. Use of nucleic acids of known copy number orconsistent copy number allows quantitating the copy number of targetnucleic acids in a sample. The standard may be a single copy gene, anucleic acid of known copy number, or when quantitating DNA copy number,a constitutively expressed housekeeping gene (see Johnson, M. R. Anal.Biochem. 278: 175-184 (2000); Boulay, J.-L., et al., Biotechniques 27:228-232 (1999)).

Methods

Disclosed herein are methods for determining the copy number of a firsttarget nucleic acid and a second target nucleic acid, comprising: a)contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system, b)amplifying by polymerase chain reaction the first target nucleic acidwith the first primer set to form a first amplicon having a firstmelting temperature (Tm) and amplifying by polymerase chain reaction thesecond target nucleic acid with the second primer set to form a secondamplicon having a second Tm, wherein said second Tm is higher than saidfirst Tm; c) during the polymerase chain reaction, determining theamount of the detection label at a first acquisition temperature,wherein said first signal acquisition temperature is below said firstTm; d) increasing the temperature of the reaction mixture to a secondsignal acquisition temperature and determining the amount of thedetection label, wherein said second signal acquisition temperature isabove said first Tm and below said second Tm; e) repeating steps (b)through (d) at least one time; and f) determining the relative copynumber of said first and said second target nucleic acids.

A polymerase chain reaction (PCR) is a technique to amplify (i.e.increase the copy number of) one or more copies of a target nucleic acidsequence. The amplification can be across several orders of magnitude,generating thousands to billions of copies of a particular DNA sequence.The polymerase chain reaction relies on thermal cycling, consisting ofcycles of repeated heating and cooling of the reaction to achieve DNAmelting and enzymatic replication of the nucleic acid. As PCRprogresses, the DNA generated is itself used as a template forreplication, setting in motion a chain reaction in which the DNAtemplate is exponentially amplified.

The PCR usually consists of a series of repeated temperature changescalled cycles; each cycle typically consists of 2-3 discrete temperaturesteps. PCR can be carried out with cycles that have three or four steps,each at a different temperature. The cycling is often preceded by asingle high temperature (>90° C.) step called a hold, which is appliedin order to fully melt (i.e. render single-stranded) a double-strandedtarget nucleic acid sequence, followed by a repeated set of temperaturechanges during which amplification of the target nucleic acid occurs,followed by a final hold at the end for final product extension or briefstorage. The temperatures used and the length of time they are appliedin each cycle depend on a variety of parameters. These include theenzyme used for DNA synthesis (e.g. the DNA polymerase), theconcentration of divalent ions and dNTPs in the reaction, the meltingtemperature of the primers, and the melting temperatures of the productsof amplification.

PCR includes at least a denaturation step, an annealing step and anelongation step. The elongation step can also be referred to as anextension step. In PCR the denaturing, annealing, and elongation stepsoccur, in order, at least once (a.k.a a single “cycle”), but aretypically repeated for up to 40 cycles. When the DNA polymerase usedrequires heat activation, an additional step, called the initializationstep, precedes the cycling stage of the PCR. Each step has a respectivetemperature associated with it. The temperature associated with eachstep is referred to as an initialization temperature, a denaturationtemperature, an annealing temperature, and an extension or elongationtemperature, respectively.

An initialization step can consist of heating the reaction to aninitialization temperature of 90, 91, 92, 93, 94, 95, 96, 97, or 98° C.,which can be held for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 minutes. The initialization step is typically only required when theDNA polymerases being used in the PCR requires heat activation. Forexample, if a thermostable polymerase is being used, an Initializationstep with an initialization temperature of 98° C. can be used.

A denaturation step is typically the first step in the repeating cycleof the PCR and consists of heating the reaction to a denaturationtemperature of 90, 91, 92, 93, 94, 95, 96, 97, or 98° C. for 15 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or35 seconds. The denaturation step melts the DNA template by disruptingthe hydrogen bonds between complementary bases, yielding single strandsof DNA.

An annealing step is typically the second step in the repeating cycle ofthe PCR and consists of lowering the temperature to an annealingtemperature of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70° C. for 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, or 45 seconds allowing annealing of the primers in a primer setto hybridize with a target nucleic acid. The annealing temperature canbe about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10° C. below the Tm of theprimers used. Stable DNA-DNA hydrogen bonds are formed when the primersequence very closely matches the template sequence. The polymerasebinds to the primer-template hybrid and begins DNA synthesis.

The extension/elongation step is the step where the nucleic acidpolymerase synthesizes a new nucleic acid strand complementary to thetarget nucleic acid strand by adding dNTPs that are complementary to thetarget nucleic acid in 5′ to 3′ direction, condensing the 5′-phosphategroup of the dNTPs with the 3′-hydroxyl group at the end of the nascent(extending) target nucleic acid strand. The extension time depends bothon the nucleic acid polymerase used and on the length of the targetnucleic acid to be amplified. As a rule-of-thumb, at its optimumtemperature, the nucleic acid polymerase will polymerize a thousandbases per minute. Under optimum conditions, i.e., if there are nolimitations due to limiting substrates or reagents, at each extensionstep, the amount of target nucleic acid is doubled, leading toexponential (geometric) amplification of the specific target nucleicacid. The elongation temperature at this step depends on the nucleicacid polymerase used. For example; Taq polymerase has its optimumactivity temperature at 75-80° C., and commonly a temperature of 72° C.is used with this enzyme.

PCR can also comprise a final elongation step. The final elongation canbe performed at a final elongation temperature of 68, 69, 70, 71, 72,73, 74 or 75° C. for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 minutes after the last PCR cycle to ensure that any remainingsingle-stranded DNA is fully copied to make a double-stranded DNAproduct.

PCR can also comprise a signal acquisition step wherein the amount of adetection label can be determined. The signal acquisition step can becarried out during the amplification of the target sequence. In someaspects the signal acquisition step follows a denaturation step, anannealing step and an elongation steps. The signal acquisition step iscarried out at a signal acquisition temperature. The signal acquisitiontemperature can be any temperature and can be carried out at one or moretimes during PCR. When the copy number of two or more target nucleicacids are being determined as described herein, the signal acquisitiontemperature should be different for detection of the detection label ofeach amplicon. For example, the temperatures for the two or more signalacquisition temperature should be selected such that the first signalacquisition temperature is below the Tm of the first amplicon and thesecond signal acquisition temperature is above said first Tm and belowthe Tm of the second amplicon. The difference between the two or moresignal acquisition temperatures can be 3, 4, 5, 6, 7, 8, 9, or 10° C. ASignal Acquisition Step can be carried out for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 seconds at the acquisition temperature.

PCR can also comprise a final hold step. The final hold step can be at afinal hold temperature of: This step at 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15° C. for an indefinite time. The final hold step can beemployed for short-term storage of the reaction.

The polymerase chain reaction can also comprise consecutive stages ofcycles. Each consecutive stage of cycles can comprise one or more of thePCR steps described above. Each consecutive stage of cycles can also bereferred to a “cycle” of the PCR. Each consecutive stage of cycles canbe carried out under the same or different temperatures for each cycleof the PCR. A PCR can be run where the annealing temperature is changedfor one or more of the cycles of PCR. For example, the PCR can be runfor a total of 40 cycles, wherein the annealing temperature is the samefor a first stage of cycles, then the annealing temperature is raisedfor a second stage of cycles and the annealing temperature is loweredfor the third stage of cycles.

A “homogenous system” is a system wherein amplification and detection ofa target nucleic acid takes place in the same reaction. A homogenoussystem is one which generates a detectable signal during theamplification of a target sequence. By “during the amplification” ismeant that after a cycle of PCR, but prior to a subsequent cycle of PCR.“During the amplification” is also meant to mean during PCR, but priorto a final hold step.

The relative copy number can be carried out by the methods describedelsewhere herein. For example, the methods described herein can be usedto measure the amounts of telomere (T) repeat sequences in experimentalDNA samples in one set of reaction wells, and the amount of single copygenes (S) in separate wells, in comparison to a reference DNA, to yieldrelative T/S ratios that are proportional to average telomere length. Inone aspect, the T signals can be collected in early cycles, before Ssignals rise above baseline, and S signals can be collected at atemperature that fully melts the telomere product, sending its signal tobaseline. The correlation of T/S ratios can also be correlated withTerminal Restriction Fragment (TRF) lengths measured by Southern blot todetermine copy numbers.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, wherein thecopy number of the first target nucleic acid sequence is greater thanthe copy number of the second target nucleic acid sequence, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system, b)amplifying by polymerase chain reaction the first target nucleic acidwith the first primer set to form a first amplicon having a firstmelting temperature (Tm) and amplifying by polymerase chain reaction thesecond target nucleic acid with the second primer set to form a secondamplicon having a second Tm, wherein said second Tm is higher than saidfirst Tm; c) during the polymerase chain reaction, determining theamount of the detection label at a first acquisition temperature,wherein said first signal acquisition temperature is below said firstTm; d) increasing the temperature of the reaction mixture to a secondsignal acquisition temperature and determining the amount of thedetection label, wherein said second signal acquisition temperature isabove said first Tm and below said second Tm; e) repeating steps (b)through (d) at least one time; and f) determining the relative copynumber of said first and said second target nucleic acids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system, b)amplifying by polymerase chain reaction the first target nucleic acidwith the first primer set to form a first amplicon having a firstmelting temperature (Tm) and amplifying by polymerase chain reaction thesecond target nucleic acid with the second primer set to form a secondamplicon having a second Tm, wherein said second Tm is higher than saidfirst Tm; c) during the polymerase chain reaction, determining theamount of the detection label at a first acquisition temperature,wherein said first signal acquisition temperature is below said firstTm; d) increasing the temperature of the reaction mixture to a secondsignal acquisition temperature and determining the amount of thedetection label, wherein said second signal acquisition temperature isabove said first Tm and below said second Tm; e) repeating steps (b)through (d) until the detection label is determined at said secondsignal acquisition temperature; and f) determining the relative copynumber of said first and said second target nucleic acids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system, b)amplifying by polymerase chain reaction the first target nucleic acidwith the first primer set to form a first amplicon having a firstmelting temperature (Tm) and amplifying by polymerase chain reaction thesecond target nucleic acid with the second primer set to form a secondamplicon having a second Tm, wherein said second Tm is higher than saidfirst Tm; c) during the polymerase chain reaction, determining theamount of the detection label at a first acquisition temperature,wherein said first signal acquisition temperature is below said firstTm; d) increasing the temperature of the reaction mixture to a secondsignal acquisition temperature and determining the amount of thedetection label, wherein said second signal acquisition temperature isabove said first Tm and below said second Tm, wherein the amount of thedetection label is detected at said first and said second signalacquisition temperatures during each of said amplification steps; e)repeating steps (b) through (d) at least one time; and f) determiningthe relative copy number of said first and said second target nucleicacids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system, b)amplifying by polymerase chain reaction the first target nucleic acidwith the first primer set to form a first amplicon having a firstmelting temperature (Tm) and amplifying by polymerase chain reaction thesecond target nucleic acid with the second primer set to form a secondamplicon having a second Tm, wherein said second Tm is higher than saidfirst Tm; c) during the polymerase chain reaction, determining theamount of the detection label at a first acquisition temperature,wherein said first signal acquisition temperature is below said firstTm; d) increasing the temperature of the reaction mixture to a secondsignal acquisition temperature and determining the amount of thedetection label, wherein said second signal acquisition temperature isabove said first Tm and below said second Tm, wherein the differencebetween the first Tm and the second Tm is at least 4 degrees Celsius; e)repeating steps (b) through (d) at least one time; and f) determiningthe relative copy number of said first and said second target nucleicacids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system,wherein at least one of the primers in the second primer set comprises aGC-clamp at the 5′ end of the primer b) amplifying by polymerase chainreaction the first target nucleic acid with the first primer set to forma first amplicon having a first melting temperature (Tm) and amplifyingby polymerase chain reaction the second target nucleic acid with thesecond primer set to form a second amplicon having a second Tm, whereinsaid second Tm is higher than said first Tm; c) during the polymerasechain reaction, determining the amount of the detection label at a firstacquisition temperature, wherein said first signal acquisitiontemperature is below said first Tm; d) increasing the temperature of thereaction mixture to a second signal acquisition temperature anddetermining the amount of the detection label, wherein said secondsignal acquisition temperature is above said first Tm and below saidsecond Tm; e) repeating steps (b) through (d) at least one time; and f)determining the relative copy number of said first and said secondtarget nucleic acids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system,wherein at least one of the primers in said first primer set comprises a5′ sequence that includes A and T nucleotides b) amplifying bypolymerase chain reaction the first target nucleic acid with the firstprimer set to form a first amplicon having a first melting temperature(Tm) and amplifying by polymerase chain reaction the second targetnucleic acid with the second primer set to form a second amplicon havinga second Tm, wherein said second Tm is higher than said first Tm; c)during the polymerase chain reaction, determining the amount of thedetection label at a first acquisition temperature, wherein said firstsignal acquisition temperature is below said first Tm; d) increasing thetemperature of the reaction mixture to a second signal acquisitiontemperature and determining the amount of the detection label, whereinsaid second signal acquisition temperature is above said first Tm andbelow said second Tm; e) repeating steps (b) through (d) at least onetime; and f) determining the relative copy number of said first and saidsecond target nucleic acids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system,wherein the 3′ ends of the primers of the first primer set arecomplementary to each other and wherein one primer of the first primerset is a mismatch primer comprising at least one mismatched nucleotideadjacent to the 3′ end of the primer, wherein said nucleotide is notcomplementary to the target nucleic acid, but is complementary to the 3′terminal nucleotide of the other primer in the first primer set. b)amplifying by polymerase chain reaction the first target nucleic acidwith the first primer set to form a first amplicon having a firstmelting temperature (Tm) and amplifying by polymerase chain reaction thesecond target nucleic acid with the second primer set to form a secondamplicon having a second Tm, wherein said second Tm is higher than saidfirst Tm; c) during the polymerase chain reaction, determining theamount of the detection label at a first acquisition temperature,wherein said first signal acquisition temperature is below said firstTm; d) increasing the temperature of the reaction mixture to a secondsignal acquisition temperature and determining the amount of thedetection label, wherein said second signal acquisition temperature isabove said first Tm and below said second Tm; e) repeating steps (b)through (d) at least one time; and f) determining the relative copynumber of said first and said second target nucleic acids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system,wherein one of the primers of the first primer set is blocked frompriming the first target nucleic acid b) amplifying by polymerase chainreaction the first target nucleic acid with the first primer set to forma first amplicon having a first melting temperature (Tm) and amplifyingby polymerase chain reaction the second target nucleic acid with thesecond primer set to form a second amplicon having a second Tm, whereinsaid second Tm is higher than said first Tm; c) during the polymerasechain reaction, determining the amount of the detection label at a firstacquisition temperature, wherein said first signal acquisitiontemperature is below said first Tm; d) increasing the temperature of thereaction mixture to a second signal acquisition temperature anddetermining the amount of the detection label, wherein said secondsignal acquisition temperature is above said first Tm and below saidsecond Tm; e) repeating steps (b) through (d) at least one time; and f)determining the relative copy number of said first and said secondtarget nucleic acids.

Also disclosed herein are methods for determining the copy number of afirst target nucleic acid and a second target nucleic acid, comprising:a) contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system,wherein the detection label is an intercalating dye b) amplifying bypolymerase chain reaction the first target nucleic acid with the firstprimer set to form a first amplicon having a first melting temperature(Tm) and amplifying by polymerase chain reaction the second targetnucleic acid with the second primer set to form a second amplicon havinga second Tm, wherein said second Tm is higher than said first Tm; c)during the polymerase chain reaction, determining the amount of thedetection label at a first acquisition temperature, wherein said firstsignal acquisition temperature is below said first Tm; d) increasing thetemperature of the reaction mixture to a second signal acquisitiontemperature and determining the amount of the detection label, whereinsaid second signal acquisition temperature is above said first Tm andbelow said second Tm; e) repeating steps (b) through (d) at least onetime; and f) determining the relative copy number of said first and saidsecond target nucleic acids.

In some aspects of the methods described herein, the copy number of thefirst and the second target nucleic acids measures the relative amountof the first nucleic acid as compared to the second nucleic acid.

In some aspects Target and reference sequences can be accuratelyquantitated in a multiplex QPCR using SYBR Green I as the onlyfluorescent dye and QPCR machines equipped only for single colordetection, by a strategy that prevent the amplification of the earlieramplifying template from interfering with the amplification of the lateramplifying template. In some aspects, to quantitate two templates in aset of DNA samples by MMQPCR, two requirements must be met. First, PCRconditions must be found such that, for each DNA sample in the set, whenthe cycle threshold for the earlier-amplifying product is reached, theamplification signal from the later-amplifying product is still atbaseline. Second, the later-amplifying product must have a highermelting temperature than the earlier-amplifying product, so that thelater-amplifying product's fluorescence can be monitored at atemperature low enough to keep it double-stranded, but high enough tocompletely melt the earlier-amplifying product, sending its fluorescencesignal to baseline. By designing primers to keep both PCR productssmall, and adding GC-rich 5′ tags such as GC-clamps to the primers forthe later-amplifying product, one can ensure that the later-amplifyingproduct will have the higher melting temperature.

The methods described herein can be used to quantitate the levels of twodifferent templates in a biological sample, where each template variesin copy number, and yet there is no overlap in the ranges of copy numberof the first and second templates. For example, cells have far morecopies of telomere repeats than copies of single copy nuclear genes; thesame situation applies for mtDNA copies vs. single copy genes, rDNAcopies vs. single copy genes, Alu DNA copies vs. single copy genes, etc.Similarly, in multiplex reverse transcriptase QPCR (RT-QPCR) studies ofmRNA levels, the aim often is to quantify the levels of two differentmRNA species, each varying in copy number, but with non-overlappingranges of copy number. For each of these pairs of templates, the C_(t)for the more abundant template can be collected when the amplificationsignal from the less abundant template is at baseline; and the C_(t) forthe less abundant template can be collected at a high temperature thatleaves its GC-rich product double-stranded, while completely melting andeliminating the signal from the product of the abundant template. Bycollecting fluorescence signals at two different temperatures throughoutthe PCR cycling, and analyzing those signals separately, one canquantitate each of the two templates independently with a single,monochrome detection label.

To date, it has been presumed impossible to determine the relative copynumbers of two different DNA sequences in a multiplex quantitativepolymerase chain reaction using a single DNA-intercalating dye, becausethe accumulating fluorescent signal arises from both amplicons. Themethods described herein present a strategy that allows the signals fromthe two amplicons to be collected separately. The cycle thresholds(C_(t)s) for the first amplicon are collected at earlier cycles, whenthe signal from the second amplicon is still at baseline. The C_(t)s forthe second amplicon are collected at a temperature well above themelting temperature (T_(m)) of the first amplicon, rendering the firstamplicon single-stranded and sending its signal to baseline. Primers aredesigned to make both amplicons small, and the second amplicon GC-rich,raising its T_(m). Pairs of templates that occur in biological samplesas high and low abundance species with no overlap in copy number rangesare natural targets for this approach. Even two templates with similarcopy numbers can be distinguished by applying primer and thermal cyclingdesigns that delay the amplification of one amplicon. The methodsdescribed herein can be used to determine the relative telomere lengthsin human DNA samples.

Similar Copy Number Methods

Also disclosed herein are methods determining the copy number of a firsttarget nucleic acid and a second target nucleic acid, wherein the copynumber of the first target nucleic acid sequence is similar to the copynumber of the second target nucleic acid sequence comprising: a)contacting a first target nucleic acid with a first primer set and asecond target nucleic acid, with a second primer set and adding a singledetection label to form a reaction mixture in a homogenous system, b)amplifying by polymerase chain reaction the first target nucleic acidwith the first primer set to form a first amplicon having a firstmelting temperature (Tm) and amplifying by polymerase chain reaction thesecond target nucleic acid with the second primer set to form a secondamplicon having a second Tm, wherein said second Tm is higher than saidfirst Tm; c) during the polymerase chain reaction, determining theamount of the detection label at a first acquisition temperature,wherein said first signal acquisition temperature is below said firstTm; d) increasing the temperature of the reaction mixture to a secondsignal acquisition temperature and determining the amount of thedetection label, wherein said second signal acquisition temperature isabove said first Tm and below said second Tm; e) repeating steps (b)through (d) at least one time; and f) determining the relative copynumber of said first and said second target nucleic acids.

When the copy number of the first target nucleic acid sequence issimilar to the copy number of the second target nucleic acid sequencethe cycles of the PCR or the individual steps of the PCR can be alteredor changed. For example, when the copy number of the first targetnucleic acid sequence is similar to the copy number of the second targetnucleic acid sequence the polymerase chain reaction of this method canfurther comprise at least three consecutive stages, wherein the firststage of the polymerase chain reaction comprises a polymerase chainreaction cycle wherein the annealing temperature of the polymerase chainreaction is higher than the annealing temperature of the second stage,wherein the second stage of the polymerase chain reaction comprises apolymerase chain reaction cycle wherein the annealing temperature of thepolymerase chain reaction is lower than the annealing temperature of thefirst stage, and wherein the third stage of the polymerase chainreaction comprises a polymerase chain reaction wherein the annealingtemperature of the polymerase chain reaction is lower than the annealingtemperature of the first stage and higher than the annealing temperatureof the second stage.

In some aspects, when consecutive stages of the polymerase chainreaction are employed, only the second amplicon is formed during thefirst stage of the polymerase chain reaction. In some aspects, whenconsecutive stages of the polymerase chain reaction are employed onlythe first amplicon is formed during the second stage of the polymerasechain reaction. In some aspects, when consecutive stages of thepolymerase chain reaction are employed both the first and secondamplicons are formed during the third stage of the polymerase chainreaction. In some aspects, when consecutive stages of the polymerasechain reaction are employed only the second amplicon is formed duringthe first stage of the polymerase chain reaction and only the firstamplicon is formed during the second stage of the polymerase chainreaction and both the first and second amplicons are formed during thethird stage of the polymerase chain reaction.

Primer composition and the various temperature of the PCR can also bealtered depending on the target and Tm of the primers, amplicons andpolymerases being used or generated.

In some aspects, to quantitate two templates of similar abundance byMMQPCR, the amplification of one of the templates is delayed by severalcycles. For example, one primer pair can be designed to anneal at 68°C., and the other pair at 50° C. After the initial 15 minute activationof the hot-start DNA polymerase and denaturation of the genomic DNAsample, cycling between 94° C. and 68° C. for at least four cycles wouldprovide a 4 cycle (or more) head start in amplification for the firsttemplate, leaving the second template unprimed. Next, cycling between94° C. and 50° C. for two cycles would continue amplifying the firsttemplate, but also initiate amplification from the second template. Notethat the primers for the second template can have the GC-rich 5′ tags,such as GC-clamps, to confer a high melting temperature on their PCRproduct. Since cycling between 94° C. and 50° C. for two cycles issufficient to synthesize sequences complementary to the full length ofthese primers, it follows that once those two cycles are completed, theannealing temperature can be raised again, and the remaining cycles inthe program can have a thermal profile similar to that of Stage 3 in theprotocol below (see Materials and Methods), during which thefluorescence signal is collected at two different temperatures in eachcycle.

In another aspect, a second approach to delaying the amplification ofone template by several cycles is provided to allow both primer pairs toinitiate product formation; then to apply four or more cycles with adenaturation temperature high enough to melt the low-melting ampliconbut not high enough to melt the high-melting amplicon; and finally toswitch to a thermal profile similar to that of Stage 3 (see Materialsand Methods) for the remaining cycles. The stage of cycling with alowered denaturation temperature may still allow a linear amplificationfrom the original template, which amplification was intended to bedelayed. A solution to this problem is to use both primer pairs having arelatively low initial annealing temperature, e.g. 50° C., but highersubsequent annealing temperatures for the PCR amplicons, conferred by 5′tags, such as GC-clamps. By cycling between 94° C. and 50° C. for twocycles, a sufficient to generate the initial PCR products, after whichthe annealing temperature would be raised enough to prevent any furtherpriming of the original DNA templates.

In one aspect the first target nucleic acid is in a first nucleic acidand said second target nucleic acid is in a second nucleic acid. In thisaspect the copy number of the first and the second target nucleic acidsmeasures the relative amount of said first nucleic acid as compared tosaid second nucleic acid. In another aspect, the first target nucleicacid is in a first nucleic acid and the second target nucleic acid is ina second nucleic acid. The copy number of the first target nucleic acidmeasures non tandem repeats of the first target nucleic acid in thefirst nucleic acid where the non tandem repeats are independentlyamplified by said first primer pair.

Also disclosed are methods for determining the copy number of a firsttarget nucleic acid as compared to the copy number of a second targetnucleic acid by multiplex quantitative PCR. The first target nucleicacid comprises tandem repeats and the copy number of said first targetnucleic acid sequence is greater than the copy number of said secondtarget nucleic acid sequence. The method comprises: (1) contacting asample comprising the first target nucleic acid and the second targetnucleic acid with a first primer set, a second primer set and anintercalating dye, wherein the first primer set is capable of PCRamplifying the first target nucleic acid to form a first amplicon havinga first melting temperature (Tm), wherein a first primer of the firstprimer pair comprises a mismatched nucleotide at other than the 3′terminus that does not base pair with a nucleotide in a first strand ofsaid first target nucleic acid when said first primer is hybridized tosaid first strand and wherein the second primer of said first primerpair has a 3′ nucleotide that does not base pair with a nucleotide inthe second strand of said first nucleic acid but does base pair withsaid mismatched nucleotide in a PCR transcript of said first strand whensaid second primer is hybridized to said PCR transcript, whereby thefirst amplicon produced by repeated PCR cycles has a defined size withsaid first Tm and wherein said second primer set is capable ofamplifying said second target nucleic acid to form a second ampliconhaving a second Tm, where said second Tm is greater than said first Tm;(2) PCR cycling the sample through a temperature profile that includesfirst and second signal acquisition temperatures for measuringintercalation of the dye in the first and second amplicons, where thefirst signal acquisition temperature is below the first Tm and saidsecond signal acquisition temperature is above the first Tm and belowthe second Tm; (3) repeating the PCR cycling step; and (4) measuring theintercalation signal from the intercalation of the dye at the first andsaid second signal acquisition temperatures during at least twodifferent PCR cycles to determine the relative copy number of the firstand the second target nucleic acids.

Also disclosed are methods for determining the copy number of a firsttarget nucleic acid as compared to the copy number of a second targetnucleic acid by multiplex quantitative PCR, where the copy number of thefirst target nucleic acid sequence is similar to the copy number of thesecond target nucleic acid sequence. The method comprises: (1)contacting a sample comprising the first target nucleic acid and thesecond target nucleic acid with a first primer set, a second primer setand an intercalating dye, wherein the first primer set is capable ofamplifying the first target nucleic acid to form a first amplicon havinga first melting temperature (Tm) and wherein the hybridization complexbetween the first primer pair and the first target nucleic acid has afirst primer Tm and wherein the second primer set is capable ofamplifying the second target nucleic acid to form a second ampliconhaving a second Tm and wherein the hybridization complex between saidsecond primer pair and said second target nucleic acid has a secondprimer Tm, where said second Tm is greater than said first Tm andwherein said first primer Tm is greater than said second primer Tm; (2)subjecting said sample to a predetermined number of PCR cycles whereinthe primer annealing temperature during said predetermined PCR cycles isabove said second primer Tm to prevent amplification of said secondtarget nucleic acid; (3) PCR cycling said sample through a temperatureprofile wherein the primer annealing temperature is at or below saidsecond primer Tm whereby said first and said second target nucleic acidsare PCR amplified and wherein said temperature profile includes firstand second signal acquisition temperatures for measuring intercalationof said dye in said first and second amplicons, where said first signalacquisition temperature is below said first Tm and said second signalacquisition temperature is above said first Tm and below said second Tm;(4) repeating said PCR cycling step; and (5) measuring the intercalationsignal from the intercalation of said dye at said first and said secondsignal acquisition temperatures during at least two different PCRcycles, wherein the relative copy number of said first and said secondtarget nucleic acids is determined from said intercalation signals andsaid predetermined number of PCR cycles.

Also disclosed are methods for determining the copy number of a firsttarget nucleic acid as compared to the copy number of a second targetnucleic acid by multiplex quantitative PCR, wherein the copy number ofsaid first target nucleic acid sequence is similar to the copy number ofsaid second target nucleic acid sequence. The method comprises: (1)contacting a sample comprising said first target nucleic acid and saidsecond target nucleic acid with a first primer set, a second primer setand an intercalating dye, wherein said first primer set is capable ofamplifying said first target nucleic acid sequence to form a firstamplicon having a first melting temperature (Tm) and wherein thehybridization complex between said first primer pair and said firsttarget nucleic acid has a first primer Tm and said second primer set iscapable of amplifying said second target nucleic acid to form a secondamplicon having a second Tm and wherein the hybridization complexbetween said second primer pair and said second target nucleic acid hasa second primer Tm, where said second Tm is greater than said first Tmand wherein said first primer Tm is greater than said second primer Tm;(2) subjecting said sample to a first predetermined number of PCR cycleswherein the primer annealing temperature during said predetermined PCRcycles is at or below said first primer Tm to amplify said first andsaid second target nucleic acids; (3) subjecting said sample to a secondpredetermined number of PCR cycles wherein the denaturation temperatureis below said second Tm to prevent further amplification of said secondtarget nucleic acid; (4) PCR cycling said sample through a temperatureprofile wherein the primer annealing temperature is at or below saidsecond primer Tm whereby said first and said second target nucleic acidsare PCR amplified and wherein said temperature profile includes firstand second signal acquisition temperatures for measuring intercalationof said dye in said first and second amplicons, where said first signalacquisition temperature is below said first Tm and said second signalacquisition temperature is above said first Tm and below said second Tm;(5) repeating said PCR cycling step; and (6) measuring the intercalationsignal from the intercalation of said dye at said first and said secondsignal acquisition temperatures during at least two different PCRcycles, wherein the relative copy number of said first and said secondtarget nucleic acids is determined from said intercalation signals andsaid second predetermined number of PCR cycles.

In some aspects of the methods described herein a second sample can beused as a reference for measuring copy number. The methods furthercomprise at least one reference sample comprising a reference nucleicacid comprising known copy numbers of said first and said second targetnucleic acids, said first and said second primer pairs and saidintercalating dye; wherein said second sample is subjected to the samePCR conditions as said first sample and wherein the intercalationsignals at said first and said second signal acquisition temperaturesfor said second sample are compared to the intercalation signals at saidfirst and second signal acquisition temperatures in said first sample toprovide an indication of the absolute copy number of said first and saidsecond target nucleic acids.

In some aspects the methods comprise: (1) contacting a sample comprisingsaid first target nucleic acid with a first primer set and said secondtarget nucleic acid with a second primer set, wherein said first primerset is capable of amplifying said first target nucleic acid to form afirst amplicon having a first melting temperature (Tm) and said secondprimer set is capable of amplifying said second target nucleic acid toform a second amplicon having a second Tm, where said second Tm issufficiently greater than said first Tm to ensure that said firstamplicon is completely melted at a temperature at which said secondamplicon has not yet begun to melt; in the presence of any singledetection label that fluoresces upon intercalation into double-strandedDNA; (2) repeated PCR cycling said sample through a temperature profilethat includes first and second signal acquisition temperatures formeasuring intercalation of said dye in said first and second amplicons,where said first signal acquisition temperature is sufficiently belowsaid first Tm that said first amplicon has not yet begun to melt andsaid second signal acquisition temperature is sufficiently above saidfirst Tm that the first amplicon is completely melted and sufficientlybelow said second Tm that the second amplicon has not yet begun to melt;and (3) measuring the intercalation signal from said dye at said firstand said second signal acquisition temperatures during at least twodifferent PCR cycles to determine the relative copy number of said firstand said second target nucleic acids, under a set of conditions thatallows said first signal to cross the threshold of detection at a cyclenumber at which said second signal is still at baseline.

Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for determiningthe copy number of one or more target nucleic acids, the kit comprisingone or more reagent compositions and one or more components or reagentsfor determining the copy number of one or more target nucleic acids. Forexample, the kits can include one or more reagent compositions and oneor more primer sets, a detection label, a nucleic acid polymerase, or acombination. Another form of kit can comprise a plurality of reagentcompositions. The kits also can contain, for example, nucleotides,buffers, ligase, open circle probes, gap oligonucleotides, or acombination.

Disclosed are kits that can be used in such methods. The kits caninclude at least first and second PCR primer pairs for amplifying thefirst and second target nucleic acids. Such components can be in a firstcontainer that is adapted to be used in a PCR amplification machine. Inone aspects, the test sample is added to the container and PCRamplification is carried out according to the disclosed methods. Thekits can also include one or more, or all, of the components forcarrying out PCR including deoxynucleotide triphosphates, thermostableDNA polymerase and a detection label.

In one aspect, the kit can comprise a second container, also adapted foruse in a PCR machine, containing a second sample comprising a referencenucleic acid comprising known copy numbers of the first and said secondtarget nucleic acids and optionally the other components needed forcarrying out PCR, including the first and second primer pairs and anintercalating dye. This second container is subjected to the same PCRconditions as the first container sample. The second container providesreference intercalation signals for known copy numbers of the first andsecond target nucleic acids thereby facilitating the determination ofthe absolute copy number of first and said second target nucleic acidsin a test sample. Additional containers containing reference nucleicacids having different copy numbers of the first and second targetnucleic acids can also be included in the kit. Such containers provideadditional intercalation signals that provide different reference pointsover a range of absolute copy number ratios. Such additional containersare particularly useful when the copy number of the first and secondtarget nucleic acids in a test sample can vary over a wide range.

Also disclosed are kits having a first container, adapted for use in aPCR machine, where said first container comprises a reference nucleicacid comprising known copy numbers of the first and second targetnucleic acids. The kit may further comprise at least one additionalcontainer comprising a second reference nucleic acid having a differentcopy number of the first and second target nucleic acids as compared tothe reference nucleic acid of said first container. The containersoptionally contain an intercalating dye and the other components neededfor PCR. Such kits are useful in standardizing the intercalation signalsfrom a PCR machine for one or more different reference nucleic acids.

Systems

Disclosed are systems useful for performing, or aiding in theperformance of, the disclosed method. Also disclosed are systems forproducing reagent compositions. Systems generally comprise combinationsof articles of manufacture such as structures, machines, devices, andthe like, and compositions, compounds, materials, and the like. Suchcombinations that are disclosed or that are apparent from the disclosureare contemplated. For example, disclosed and contemplated are systemscomprising solid supports and reagent compositions.

Data Structures and Computer Control

Disclosed are data structures used in, generated by, or generated from,the disclosed method. Data structures generally are any form of data,information, and/or objects collected, organized, stored, and/orembodied in a composition or medium. A target fingerprint stored inelectronic form, such as in RAM or on a storage disk, is a type of datastructure.

The disclosed method, or any part thereof or preparation therefore, canbe controlled, managed, or otherwise assisted by computer control. Suchcomputer control can be accomplished by a computer controlled process ormethod, can use and/or generate data structures, and can use a computerprogram. Such computer control, computer controlled processes, datastructures, and computer programs are contemplated and should beunderstood to be disclosed herein.

EXAMPLES Example 1 Research Subjects

Genomic DNA was extracted directly from blood samples by standardprocedures, and stored long-term in TE⁻⁴ (10 mM Tris-HCl, 0.1 mM EDTA,pH 7.5) at 4° C. at a concentration of approximately 100 ng permicroliter. DNA stocks were diluted into pure water just prior tosetting up QPCR runs. The samples, from 95 Utah individuals (47 femalesand 48 males, age range 5-94 years), are those analyzed in our previouspaper describing telomere length measurement by monoplex quantitativePCR (Cawthon, R. M. (2002) Telomere measurement by singleplexquantitative PCR. Nucleic Acids Res, 30, e47).

Monochrome Multiplex Quantitative PCR (MMQPCR)

PCR reactions were set up by aliquoting 15 microliters of master mixinto each reaction well of a 96-well plate compatible with the Bio-RadMyiQ Single Color Real-Time PCR Detection System, followed by 10microliters of each experimental DNA sample, containing approximately 20nanograms of DNA diluted in pure water, for a final volume of 25microliters per reaction. Five concentrations of a reference DNA sample(the “Standard DNA”) spanning an 81-fold range of DNA concentration wereprepared by serial dilution and analyzed in duplicate in every 96-wellplate in this study; these reactions provided the data for thegeneration of the standard curves used for relative quantitation. Allexperimental DNA samples were assayed in triplicate.

The final concentrations of reagents in the PCR were 0.75×SYBR Green I(Invitrogen), 10 mM Tris-HCl pH 8.3, 50 mM KCl, 3 mM MgCl₂, 0.2 mM eachdNTP, 1 mM DTT, and 1M betaine (U.S. Biochemicals). Each 25 microliterreaction received 0.625 U AmpliTaq Gold DNA polymerase (AppliedBiosystems, Inc.).). For multiplex QPCR, the telomere primer pair telgand telc (final concentrations 900 nM each), were combined either withthe albumin primer pair albu and albd (final concentrations 900 nMeach), or with the beta-globin primer pair hbgu and hbgd, (finalconcentrations 500 nM each) in the master mix. All primer sequences andthe rationale for their design are presented in the Results section.

The thermal cycling profile was Stage 1: 15 min at 95° C.; Stage 2: 2cycles of 15 s at 94° C., 15 s at 49° C.; and Stage 3: 32 cycles of 15 sat 94° C., 10 s at 62° C., 15 s at 74° C. with signal acquisition, 10 sat 84° C., 15 s at 88° C. with signal acquisition. The 74° C. readsprovided the C_(t)s for the amplification of the telomere template; the88° C. reads provided the C_(t)s for the amplification of the singlecopy gene template.

After thermal cycling and raw data collection were complete, the MyiQsoftware (Bio-Rad iQ5 2.0 Standard Edition Optical System Software) wasused to generate two standard curves for each plate, one for thetelomere signal and one for the scg signal. The T/S ratio for anexperimental DNA sample is T, the number of nanograms of the StandardDNA that matches the experimental sample for copy number of the telomeretemplate, divided by S, the number of nanograms of the Standard DNA thatmatches the experimental sample for copy number of the scg. As eachexperimental sample was assayed in triplicate, three T/S results wereobtained for each sample; the final reported result for a sample in agiven run is the average of the three T/S values. Average T/S isexpected to be proportional to the average telomere length per cell.Samples with a T/S>1.0 have an average telomere length greater than thatof the Standard DNA; samples with a T/S<1.0 have an average telomerelength shorter than that of the Standard DNA.

Determination of Mean Terminal Restriction Fragment (TRF) Lengths

Mean TRF lengths were determined in duplicate as described previously(Cawthon, R. M. (2002) Nucleic Acids Res, 30, e47). Briefly, DNA wasdigested with HaeIII restriction endonuclease, and digested samples weremixed with DNA size standards prior to agarose gel electrophoresis andSouthern blotting onto nylon membranes. Following hybridization of theblots with a radioactive telomeric oligonucleotide probe (TTAGGG)₇ SEQID NO:1) and capture of the telomere smear images, blots were strippedand hybridized with radioactive probes specific for the DNA sizestandards. The size standard images and telomere smear images were thensuperimposed to locate the positions of the size intervals within thetelomere smears. Mean TRF length was then calculated asΣ(OD_(i))/Σ(OD_(i)/L_(i)), where OD_(i) is total radioactivity abovebackground in interval i and L_(i) is the average length of i inbasepairs.

Results Primers that Amplify a Fixed-Length Product from TelomericTandem Hexamer Repeats

Relative average telomere length can be measured by quantitative PCRusing primers that hybridize the telomeric hexamer repeats, because thenumber of binding sites for the primers increases as average telomerelength increases. Our original tel1 and tel2 primers for telomere lengthmeasurement by singleplex QPCR (1) are both able to prime at multiplelocations along the tandem repeats of telomeric DNA. They thereforegenerate a series of products of various sizes, some of which melt attemperatures high enough to overlap the melting curve of the scg'samplicon. Consequently, “clean” reads, at a high temperature, of theSYBR Green I fluorescence signal from the scg's double-strandedamplicon, without any interfering signal from double-stranded telomerePCR products, as required for successful MMQPCR, are not possible whentel1 and tel2 are the telomere primers.

To solve this problem, a pair of telomere primers were designed, telg,ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT SEQ ID NO:2) and telc,TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA SEQ ID NO:3), that generate ashort, fixed-length product (FIG. 1). Only telg is able to prime DNAsynthesis along native telomeric DNA sequences. The telc primer isblocked from priming native telomeric DNA by a mismatched base at its 3′terminus. However, telc is able to hybridize along various stretches ofthe telg primer extension product, and exactly one configuration ofthose hybridizations allows the priming of DNA synthesis, therebyenabling the generation of a single, fixed-length product. This isachieved by introducing a nucleotide change in telg at the third basefrom the 3′ end, such that the last 3 bases of the telg and telc primersoverlap with perfect complementarity. This overlap is not sufficient toallow the native telg and telc primers to prime each other efficiently,so primer dimer formation is undetectable over the range of cycles thattelomere length quantitation occurs. However, when the telg extensionproduct is hybridized to telc, this three base overlap is the only sitewhere the 3′ end of telc can efficiently prime DNA synthesis. See e.g USPatent Publication 2003/0162266. The resulting PCR product is,therefore, of fixed length, and three bases shorter than the sum of thelengths of the two primers used to generate it. The sharp melting curvefor this product (green curve in FIG. 2) is consistent with specific,fixed-length product formation, and agarose gel electrophoresis in 6%gels revealed only the expected 79 bp product (data not shown). FIG. 2also demonstrates that the melting curve for the telomere PCR product iswell separated from the melting curve for the albumin PCR product (bluecurve in FIG. 2), allowing the SYBR Green I signal from albumin to beread at a temperature that fully melts the telomere PCR product.

Primer Design for Single Copy Genes (Albumin and Beta-Globin)

Primers were designed so that the scg amplicon would melt at a muchhigher temperature than the telomere amplicon. Fluorescent signal fromthe scg amplicon could then be acquired at a temperature high enough tocompletely melt the telomere amplicon, eliminating its contribution tothe signal, but low enough to keep the scg amplicon double-stranded andtherefore able to bind SYBR Green I.

The primers for amplification of the scg albumin are albu:CGGCGGCGGGCGGCGCGGGCTGGGCGGaaatgctgcacagaatccttg SEQ ID NO:4); and albd:GCCCGGCCCGCCGCGCCCGTCCCGCCGgaaaagcatggtcgcctgtt SEQ ID NO:5). Thepredicted product size is 98 bp. The primers for amplification of thescg beta-globin are hbgu:CGGCGGCGGGCGGCGCGGGCTGGGCGGcttcatccacgttcaccttg SEQ ID NO:6); and hbgd:GCCCGGCCCGCCGCGCCCGTCCCGCCGgaggagaagtctgccgtt SEQ ID NO:7). Thepredicted product size is 106 bp. Capitalized bases are non-templated5′tag sequences that confer a very high melting temperature on theresulting PCR product. Please note that the 5′ tag sequences for thealbumin primers are identical to those used in the beta-globin primers.Note also that the two GC-rich 5′ tagging sequences in each primer setare very different from each other; if they were the same, hairpinformation shutting down amplification would be likely to occur duringthe PCR.

The addition of a GC-clamp to the 5′ end of a PCR primer to raise themelting temperature of one end of the PCR product is common practicewhen screening a gene for point mutations by Denaturing Gradient GelElectrophoresis (2). By attaching 5′ GC-clamps to both of the primersused to amplify the scg, and keeping the targeted genomic sequenceshort, a PCR product with a very high melting temperature will begenerated. FIG. 2 shows that the T_(m) for the doubly GC-clamped albuminPCR product is above 9° C. Agarose gel electrophoresis in 6% gelsrevealed only the expected size product. Similar results were obtainedfor the doubly GC-clamped beta-globin PCR product (data not shown).

The 5′ GC-clamps also ensure that both of the primers used to amplifythe scg have T_(m)s for their amplicon that are higher than the T_(m) ofthe telomere PCR product. The benefits of this design are discussedbelow (see Thermal profile and cycling design). An analysis using theOligoAnalyzer program (www.idtdna.com) indicated that all four scgprimers (albu, albd, hbgu, and hbgd) have T_(m)s greater than 84° C. inthe buffer composition used in this study.

Thermal Profile and Cycling Design

In Stage 1 of the thermal cycling protocol, the AmpliTaq Gold DNApolymerase is heat-activated, and the genomic DNA sample is denatured.In Stage 2, two cycles of relatively low temperature are needed toeffectively anneal and extend the telomere primers, due to the presencein those primers of purposely introduced mutations that preventformation and amplification of primer dimer PCR products (1).

In Stage 3 the repeating cycle begins with a denaturation, an annealing,and an extension step with signal acquisition that are typical ofconventional QPCR. These are followed by two unconventional steps:incubation at 84° C. for 10 s, and incubation at 88° C. for 15 s with asecond signal acquisition. Heating to 84° C. melts the early-amplifyingtelomere product, releasing DNA polymerase (which binds double-stranded,but not single-stranded DNA, ref 3) for work on the scg PCR product,where DNA synthesis can proceed, due to the high annealing temperatures(above 84° C.) of the scg primers, and the ability of Taq DNA polymeraseto maintain robust activity even at 84° C. (4).

In conventional multiplex PCR, high concentrations of the earliestamplifying product often inhibit subsequent amplification of lessabundant templates, due to the above-mentioned binding up of DNApolymerase by the early product. The usual recommended solution is tolimit the primer concentrations for the more abundant target sequence,so that less product is formed, leaving enough DNA polymerase unboundand free to continue copying the less abundant template. But loweringprimer concentrations often results in a reduced PCR efficiency, or evena complete failure to amplify the target sequence. Reduced efficienciesalso contribute to greater variation in C_(t) values between replicates.The 84° C. incubation step in MMQPCR eliminates the need to limit theprimer concentrations for the more abundant template, releasingpolymerase from even high concentrations of the corresponding PCRproduct, so that the second product can be synthesized efficiently.

Heating further to 88° C. for the second signal acquisition step ensuresthat the telomere PCR product is completely melted and unable tointerfere with the collection of the rising SYBR Green I fluorescencesignal from the accumulating scg amplicon.

Validity of the MMQPCR Method Over the Natural Range of Telomere Lengths

FIG. 3 shows amplification curves collected at two differenttemperatures (74° C. and 88° C.) for three reference human genomic DNAsamples previously shown to have high, middle, or low average telomerelengths (approximately a 3-fold range of telomere lengths). Based on themelting profiles presented in FIG. 2, the 74° C. reads should detectboth telomere and albumin PCR products, and the 88° C. reads shoulddetect only the albumin product. However, because the albumin templateis much lower in copy number than the telomere template in each DNAsample, the 74° C. C_(t)s, all collected when the corresponding albuminsignals were still at baseline, are measures of telomere amplificationonly. (It has been confirmed, in reactions without the telomere primers,that the single copy gene signal rises above baseline at essentially thesame cycle number whether collected at 74° C. or 88° C.) Even the samplewith the shortest telomeres (approximately 1,670 bp), and therefore themost right-shifted amplification curve (blue curve), crosses thresholdat a cycle number when the albumin gene's amplification signal is stillat baseline. In the present study of 95 whole blood DNA samples fromsubjects aged 5-94 years, each sample's scg amplification signal was atbaseline when the C_(t) for the corresponding telomere signal wascollected.

Independent Standard Curves for Telomere and Single Copy Gene

FIG. 4 shows two independent standard curves, one for the telomererepeats and another for the scg albumin, determined for the Standard DNAby acquiring the SYBR Green I fluorescence signal at two differenttemperatures (74° C. for the telomere signal, and 88° C. for the albuminsignal) in each cycle of Stage 3 of the cycling protocol. This same DNAsample was used to generate two standard curves for each separate PCRreaction plate in this study. In this semi-log plot of DNA concentrationvs. cycle threshold, both curves are linear over the 81-fold DNAconcentration range. The PCR efficiencies for both telomere and albuminamplifications were greater than 90%, and approximately equal. For thisparticular Standard DNA sample, at each DNA concentration the C_(t) foralbumin occurred approximately six cycles later in cycling than theC_(t) for the telomere repeats.

In FIG. 3, essentially identical amounts of DNA were inputted into thereactions (based on OD₂₆₀ UV spectrophotometer readings), so that thedifferences in C_(t) observed at 74° C. would reflect only differencesin telomere length (without influence from variation in the amounts ofinput DNA). (In normal practice, there is no need to precisely matchexperimental samples for input DNA, since the procedure of normalizingthe T signal to the S signal addresses this issue. A wide range of inputDNA amounts are acceptable, as long as both T and S signals fall withinthe range of the T and S standard curves; see FIG. 4.) The nearlyperfect overlap of the three amplification curves acquired at 88° C. isexpected, since only the single copy gene (albumin gene) signal iscollected at this temperature. The bottom panel shows that the cyclethresholds for the telomere signals can be collected at 74° C. when thealbumin signal is still at baseline. (It has been confirmed, inreactions without the telomere primers, that the single copy gene signalrises above baseline at essentially the same cycle number whethercollected at 74° C. or 88° C. Also, it has been confirmed, in reactionswithout the single copy gene primers, that the telomere amplificationsignal is completely flat and at zero throughout the PCR run when readat 88° C., as would be expected based on the melting profiles shown inFIG. 2.) Since the Bio-Rad MyiQ software can display only onetemperature's amplification curves at a time, the displays for the 74°C. and 88° C. reads have been superimposed.

Correlation Between Mean TRF Lengths and Relative T/S Ratios

To test the validity of the MMQPCR approach to telomere lengthmeasurement, the relative telomere lengths (average T/S ratios) in wholeblood DNA samples from 95 individuals, aged 5-94 years, measured intriplicate by MMQPCR, were compared to the mean Terminal RestrictionFragment (TRF) lengths of these same DNA samples as measured by thetraditional Southern blot approach (1). FIG. 5 shows the strongcorrelation in relative telomere lengths measured by these verydifferent techniques (R²=0.844). This correlation is higher than thecorrelation as reported previously (1) for T/S ratios measured in thesesame samples by singleplex QPCR vs. their mean TRF lengths (R²=0.677).

Reproducibility of T/S Ratio Measurements

To examine the intra-assay reproducibility of T/S measurements byMMQPCR, the coefficient of variation (standard deviation divided by themean) for T/S was determined for each of the 95 DNA samples assayed intriplicate in a single run of the MMQPCR assay, using albumin as thescg. The intra-assay geometric mean of the coefficient of variation was5.22%. To examine inter-assay reproducibility, the measurements of T/Sin the same 95 DNA samples was repeated, again in triplicate, on anotherday, taking care that the specific MyiQ PCR machine and reaction wellpositions occupied by each DNA sample were different in these twoindependent runs of the assay. FIG. 6 shows the strong correlationbetween the average T/S ratios determined by the first and second runs(R²=0.91). The slope of the linear regression line through the data wasnear unity, and the y-intercept near zero, as expected. The coefficientof variation for each of the 95 pairs of average T/S values from the twoindependent runs was determined. The inter-assay geometric mean of thecoefficient of variation was 3.13%.

T/S Ratios are Independent of the Single Copy Gene Used

To test whether using beta-globin, instead of albumin, as the scg mightalter apparent relative telomere lengths, the measurements of T/S in thesame 95 DNA samples were repeated in triplicate, in two separate runs,substituting the beta-globin primers for the albumin primers. FIG. 7plots the average T/S values from the two runs with albumin as the scg(x axis) vs. the average T/S values from the two runs with beta-globinas the scg (y axis). The T/S values obtained with albumin agreecorrelated highly with those obtained using beta-globin (R²=0.934).

Relative telomere lengths (T/S ratios) measured in 95 DNA samples by themonochrome multiplex quantitative PCR method described were very highlycorrelated with relative Terminal Restriction Fragment lengths measuredby Southern blot. The T/S ratios measured in these same samples by anoriginal singleplex QPCR assay were not as highly correlated with theTRF lengths. These results suggest that telomere length measurement byMMQPCR is more accurate than telomere length measurement by singleplexQPCR. Furthermore, T/S results obtained by MMQPCR were highlyreproducible in independent runs of the assay. Multiplexing the telomereQPCR assay allows increased throughput and lower costs for epidemiologicstudies of telomere length. Furthermore, the usual additional costassociated with converting to a multiplex assay, of having to synthesizeor purchase expensive custom-made multi-color fluorescent probes is alsoavoided by adopting this method.

MMQPCR can be easily adapted for the study of many pairs of DNAtemplates that naturally occur at very different copy numbers, e.g.mtDNA copies vs. single copy genes, rDNA copies vs. single copy genes,and Alu DNA copies vs. single copy genes. Similarly, pairs of RNAspecies with very different copy numbers may be quantifiable by thismethod, following reverse transcription into cDNA. For most pairs oftargets, standard principles of primer design can be followed, with theonly additional guidelines being that the primers for the more abundanttemplate may generate a relatively short product (40-80 bp) so that itsT_(m) will be appropriately low (<83° C.), and the primers for the lessabundant template may contain the 5′ GC-clamps presented here (orsimilar ones) and generate a short product so that its T_(m) will besufficiently high (>90° C.). Furthermore, the nuisance and attendantdifficulties, in conventional multiplex QPCR, of having to limit theprimer concentrations used to amplify the more abundant template, iseliminated in MMQPCR. The design features of the telg and telc telomereprimers herein were used for amplifying short tandem repeats withprimers that hybridize to those repeats. In addition to measuringtelomere lengths by MMQPCR, mtDNA to nDNA ratios were measured by thisapproach, and it worked well. Even pairs of templates with similar copynumbers may be studied by this approach by applying primer and thermalprofile designs that delay the amplification of one amplicon.

Example 2 MMQPCR of Two Targets of Similar Abundance

In the event that two target nucleic acids are in similar abundance,MMQPCR can be employed. In order to do so, one artificially delays theamplification of one target, while allowing the other target to continueto amplify. For example, the compositions and methods described inExample 1 are used to determine the copy number of a first targetnucleic acid and the copy number of a second nucleic acid, when thefirst and second target nucleic acids are similar in abundance. To doso, one uses the same compositions and methods described above, exceptfor the PCR cycling parameters provided. A thermal cycling profile couldbe Stage 1: 15 min at 95° C.; Stage 2: 2 cycles of 15 s at 94° C., 15 sat 49° C.; Stage 3: 2 to 6 cycles of 15 s at 88° C., 10 s at 62° C., 15s at 74° C.; Stage 4: 32 cycles of 15 s at 94° C., 10 s at 62° C., 15 sat 74° C. with signal acquisition, 10 s at 84° C., 15 s at 88° C. withsignal acquisition.

Stage 3 above will allow exponential amplification of the telomereproduct, because it is fully melted at 88 degrees. However, the singlecopy gene product is fully duplexed at 88 degrees, so the single copygene primers cannot get onto and amplify the single copy gene productduring this Stage 3 cycling.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are specifically incorporated by reference. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such disclosure by virtue of prior invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

I claim:
 1. A method for determining in a target sample the copy numberof a first target nucleic acid and a second target nucleic acid, themethod comprising: a) contacting a first target nucleic acid with afirst primer set and a second target nucleic acid with a second primerset and adding a single detection label to form a reaction mixture in ahomogenous system, wherein one of the primers of the first primer set isblocked from priming the first target nucleic acid and wherein one ofthe primers of the first primer set is blocked from priming the firsttarget nucleic acid; wherein the primer blocked from priming the firsttarget nucleic acid comprises a mismatched base at its 3′ end″ andwherein the first target nucleic acid comprises a tandem repentsequence; b) amplifying by polymerase chain reaction the first targetnucleic acid with the first primer set to form a first amplicon having afirst melting temperature (Tm) and amplifying by polymerase chainreaction the second target nucleic acid with the second primer set toform a second amplicon having a second Tm, wherein said second Tm ishigher than said first Tm; c) during the polymerase chain reaction,determining the amount of the detection label at a first acquisitiontemperature, wherein said first signal acquisition temperature is belowsaid first Tm; d) increasing the temperature of the reaction mixture toa second signal acquisition temperature and determining the amount ofthe detection label, wherein said second signal acquisition temperatureis above said first TM and below said second Tm; e) repeating steps (b)through (d) at least one time; and f) determining the copy number ofsaid first and said second target nucleic acids.
 2. The method of claim1, wherein the copy number of the first target nucleic acid sequence isgreater than the copy number of the second target nucleic acid sequence.3. The method of claim 1, wherein the amount of the detection label isdetected at said first and said second signal acquisition temperaturesduring each of said amplification steps.
 4. The method of claim 1wherein the difference between the first Tm and the second Tm is atleast 4 degrees Celsius.
 5. The method of claim 1, wherein at least oneof the primers in said first primer set comprises a 5′ sequence thatincludes A and T nucleotides.
 6. The method of claim 1, wherein the 3′ends of the primers of the first primer set are complementary to eachother.
 7. The method of claim 6, wherein one primer of the first primerset is a mismatch primer comprising at least one mismatched nucleotideadjacent to the 3′ end of the primer, wherein said nucleotide is notcomplementary to the target nucleic acid, but is complementary to the 3′terminal nucleotide of the other primer in the first primer set.
 8. Themethod of claim 7, wherein the extension product of the mismatch primerof the first primer set is capable of hybridizing to the other primer inthe first primer set.
 9. The method of claim 1, wherein the primerblocked from priming the first target nucleic acid comprises amismatched base at its 3′ end.
 10. The method of claim 1 wherein thedetection label is an intercalating dye.
 11. The method of claim 1,wherein the copy number of a first and a second target nucleic acidsmeasures the relative amount of the first nucleic acid as compared tothe second nucleic acid.
 12. The method of claim 1, wherein the firsttarget nucleic acid is obtained from a sample.
 13. The method of claim1, wherein the copy number of the first target nucleic acid determinesthe number of tandem repeat sequences present in the sample.
 14. Themethod of claim 1, wherein the copy number of the first target nucleicacid sequence is similar to the copy number of the second target nucleicacid sequence.
 15. The method of claim 14, wherein the polymerase chainreaction comprises at least three consecutive stages of cycles, whereinthe first stage of cycles of the polymerase chain reaction comprises apolymerase chain reaction wherein the annealing temperature of thepolymerase chain reaction is higher than the annealing temperature ofthe second stage of cycles, wherein the second stage of cycles of thepolymerase chain reaction comprises a polymerase chain reaction whereinthe annealing temperature of the polymerase chain reaction is lower thanthe annealing temperature of the first stage of cycles, and wherein thethird stage of cycles of the polymerase chain reaction comprises apolymerase chain reaction wherein the annealing temperature of thepolymerase chain reaction is lower than the annealing temperature of thefirst stage of cycles and higher than the annealing temperature of thesecond stage of cycles.
 16. The method of claim 15, wherein only thefirst amplicon is formed during the first stage of cycles of thepolymerase chain reaction.
 17. The method of claim 15, wherein only thesecond amplicon is formed during the second stage of cycles of thepolymerase chain reaction.
 18. The method of claim 15, wherein both thefirst and second amplicons are formed during the third stage of cyclesof the polymerase chain reaction.
 19. The method of claim 15, whereinthe amplification step is repeated until the detection label isdetermined at said second signal acquisition temperature.
 20. The methodof claim 15, wherein the amount of the detection label is detected atsaid first and said second signal acquisition temperatures during eachof said amplification steps.
 21. The method of claim 15, wherein thedifference between the first Tm and the second Tm is at least 4 degreesCelsius.
 22. The method of claim 15, wherein at least one of the primersin said first primer set comprises a 5′ sequence that includes A and Tnucleotides.
 23. The method of claim 15, wherein the 3′ ends of theprimers of the first primer set are complementary to each other.
 24. Themethod of claim 23, wherein one primer of the first primer set is amismatch primer comprising at least one mismatched nucleotide adjacentto the 3′ end of the primer, wherein said nucleotide is notcomplementary to the target nucleic acid, but is complementary to the 3′terminal nucleotide of the other primer in the first primer set.
 25. Themethod of claim 24, wherein the extension product of the mismatch primerof the first primer set is capable of hybridizing to the other primer inthe first primer set.
 26. The method of claim 15, wherein one of theprimers of the first primer set is blocked from priming the first targetnucleic acid.
 27. The method of claim 26, wherein the primer blockedfrom priming the first target nucleic acid comprises a mismatched baseat its 3′ end.
 28. The method of claim 15, wherein the detection labelis an intercalating dye.
 29. The method of claim 15, wherein the copynumber of a first and a second target nucleic acids measures therelative amount of the first nucleic acid as compared to the secondnucleic acid.
 30. The method of claim 15, wherein the first targetnucleic acid comprises a tandem repeat sequence.
 31. The method of claim15, wherein the first target nucleic acid is obtained from a sample. 32.The method of claim 31, wherein the copy number of the first targetnucleic acid determines the number of tandem repeat sequences present inthe sample.
 33. The method of claim 1, wherein the amount of detectionlabel determined during the first and said second signal acquisitiontemperatures are compared to a control.
 34. The method of claim 1,wherein the amount of detection label determined during the first andsaid second signal acquisition temperatures are compared to a control.